Survey Methods for Frog Abnormalities on National Wildlife Refuges

              A Presentation of the United States Fish and Wildlife Service
Survey Methods for Frog Abnormalities on National Wildlife Refuges

Narrator: Introduction

Amphibians, the group of animals that include frogs, toads, and salamanders, are found in 
forty-nine states.  Amphibians are quite sensitive to changes in their environment and can be 
considered indicators of environmental quality for several reasons.  First, during various life 
stages, they live both in the water and on land.  Their skin is highly permeable to water and air. 
Thus, they can absorb gases and other constituents, such as pollutants from their environment. 
Amphibians go through a process called metamorphosis where by they change from their larva stage 
into adults.  During this part of their life cycle, the changes in these animals are extreme, 
making them particularly vulnerable to disturbances in their environment.  For example, during 
metamorphosis, the immune system is temporarily suppressed, there by increasing the chances
for infection or disease.  Because of their sensitivity to changes in their surroundings, 
amphibians can be considered the modern day canary in a coalmine.

This training video is designed for Fish and Wildlife Service biologists and technicians who will 
be asked to conduct frog surveys primarily at National Wildlife Refuges.  The purpose of this video 
is to show the proper survey methods, handling techniques, processing and shipping methods.  The 
U.S. Fish and Wildlife Service maintains over 500 National Wildlife Refuges, which provide 
important habitat for amphibians and other resources.

In the Northeast and Midwest regions, biologists began surveying in 1997 to gather basic 
information on the occurrence of abnormal frogs on refuge lands.  In 2000, the survey was expanded 
nationwide.  The Service has worked closely with our partners including the U.S. Geological Survey, 
universities, and state agencies in developing the project, analyzing data, reporting and sharing 
our findings.

The phrases abnormality, malformation, and deformity are often used interchangeably. For our 
purposes, “abnormality” is defined as missing, extra, or unusual body parts based on field 
observations.  A malformation occurs when something goes wrong during developmental stages, causing 
an organ or body part to form improperly.  A deformity occurs when a body part that already exists 
becomes disfigured.  There are different habitats where frogs and other amphibians can be found.  
They are found at different times of the year according to your locations.  You should be familiar 
with the species and life histories of the frogs living in your area.

Although the focus of this video is for sampling on refuges, the basic skills would apply to other 
lands as well.  The most important contacts for your survey are the refuge manager and refuge 
biologist.  Talk with them early in the season and visit the refuges that possess the habitats 
where frogs are found.  Carry a species key or guide with you so that you know which species
are likely to be found in your area and what are the appropriate size ranges for metamorphs.

This is the equipment you will need and you should find this list in your training guide:
    Hip boots or waders-have both.
    Long-handled 24” deep mesh net-one per person.
    Plastic containers with tight fitting lids with holes punched in them.
    Clear plastic bag or plastic containers with tight fitting lids without holes for tadpoles.
    Data Sheets.
    Rulers (metric).
    Field Guide to Malformations of Frogs and Toads.

    Hand lens or magnifying glass.
    Field vest for holding plastic containers.
    Two-foot deep plastic container with lid for holding frogs.
    Five-gallon bucket with lid for holding frogs.
    Camera.
    GPS recorder.
    Hydro lab or similar device.

Two biologists, Fred Pinkney and Sherry Krest, will be demonstrating the techniques for capturing 
and handling frogs in this video. (0:46-5:43)

Fred: Well I’ve got all our equipment together and I think we’re just about ready to go out and 
collect some frogs.  So we’re just going to review all the equipment we’ve got and make sure we 
have everything.  You see with these long handled nets which are good, we like good deep nets so 
when we get a frog or a toad it can’t hop out on us.  Once we get them, we’ll go over the
technique once we get in the water. I like to store them in these little plastic containers – what 
we did we punched some holes in them ...  These vests are handy because -- not all you guys have 
vests -- but you can put them in your pocket and then just keep going – it doesn’t slow you
down.

Sherry: And this is another type of container you can use – prefer the smaller ones, but these will 
do too.

Fred: We’ll record the site with a GPS just so we have the location. I like to record what grid 
we’re using and then we’ll take a picture, a couple pictures, of the site.  We’ll also have the 
camera ready once we examine the frogs.  If we see anything that looks unusual we can take a 
picture now. We can also take the frogs back to the lab with us and use a better camera with the 
macro lens and try to really get some good photographs if we see something interesting.  So we’re 
going to go out and try to get at least fifty metamorph frogs of the same species and what
we’ll probably get here are green frogs.  We may get southern leopard frogs and Sherry and I can go 
over what you’ve got in case there’s any doubt.  The size we’re looking for is forty
millimeters -- snout to vent length -- so that’s about this big for the green frogs and southern 
leopard frogs.  We’re also looking for cricket frogs, which are much smaller, and the snout to vent 
length on those is 15 millimeters so they’re pretty small.  Basically in the field catch whatever 
you can; if it’s an obvious huge adult – let it go and then we’ll sit around and do the 
measurements as a group later.

Sherry: And make sure is has all four legs – it can have a tail, but all four legs.

Fred: If you happen to get tadpoles, while you’re scooping in the water, a tadpole that has both 
four limbs and hind limbs is considered a metamorph for this study, so if you have something that 
looks like a tadpole but you see four legs on it – that’s good.  If you have a tadpole, we’ll have 
a container that doesn’t have holes that you can carry with you for the tadpoles.  So the frogs 
will go in the containers with the holes, the tadpoles in the containers without the holes. Once we 
get back here, we’ll sit in a group -- probably two groups of three -- and we’ll have a recorder 
whose going to record the data on these data sheets and an examiner whose going to measure the 
frogs and we’ll go through these check lists and get everything recorded.  And if there is any 
doubt of what we’re seeing we can use this field guide that has a lot of good pictures and x-ray 
pictures of the frogs.  Once we finish, we’ll also go and use the Hydrolab to record water quality. 
 I think what will probably work best is if we start out in three groups of two.

This is Blue Gill Pond.  You can see it’s got a lot of submerged aquatic vegetation. There’s a lot 
of frogs that are going to be on the edge here and I think the best way to do this is to work in 
pairs and basically the two things you want to do is you want to try and have a quick linear
motion after the frog, not a big looping thing cause you’ll never get them.  You can work in pairs 
effectively.  Sometimes by having one person hold the net and the other person put their foot 
behind the frog and chase it into your net.  You’ll just sort of develop your own style as you
work together.  So let’s give it a try.

Metamorphs
Sherry: discusses the southern leopard frog-Rana sphenocephala; cricket frog-Acris crepitans;
gray tree frog-Hyla versicolor; green frog-Rana clamitans melanota (11:58-12:48)

Tadpoles
Narrator: Although you will be collecting metamorphs, you should also be familiar with tadpole and 
adult morphology. (12:48-12:55)

Sherry: discusses tadpoles of the green frog-Rana clamitans melanota; bull frog-Rana catebeiana; 
northern cricket frog-Acris crepitans (12:57-13:43)

Adults
Sherry: discusses adults of the green frog; southern leopard frog; cricket frog (13:48-14:25)



Measuring & Handling




































Sherry: We’ve been surveying for about an hour now and we’ve collected all of our frogs in this 
little tub and we’re going to put them in the bigger tub and it has some water in the bottom 
approximately half an inch to an inch and a cold pack to keep the frogs comfortable so they don’t 
over heat.  We’ve got several species in here. You need a tub with very high sides because as
you can see they immediately start to come to the top.

Fred: We’ve collected about fifty metamorphs from this pond and now we are going to do our 
measurement and examination of each animal. We take the frogs from our individual plastic 
containers and place them in this large rubber enclosed bucket and we have about one half inch to 
one inch of pond water in the bottom to keep the animals moist.  Now Sherry has just reached
in and collected this green frog.  She’s moistened her hands to keep them wet so she won’t injure 
the frog during handling. The first thing she’s going to do is to turn the frog over and measure
the snout to vent length.  We have operationally defined 40 millimeters as the largest size for a 
green frog metamorph.  What is the length of this one?

Sherry: 42 millimeters.
Fred: By the terms of our study this animal would not be used and would be released. This is a 
metamorph cricket frog.  These are much smaller than the green frogs and we’ve
operationally defined the maximum size for a cricket frog metamorph at 15 millimeters.  Sherry is 
now measuring the snout to vent length.

Sherry: 14 millimeters.

Fred: I record that on the data sheet. Since this still has a tail we take a separate measurement 
of the tail length.

Sherry:  21 millimeters.

Fred: She starts the examination looking at the head. Is the frog missing any eyes?
Sherry: No
Fred: Is there a small eye?
Sherry: No
Fred: Is the iris normal?
Sherry: Yes
Fred: Is there a displaced or malpositioned eye?
Sherry: No
Fred: Is the head small or domed?
Sherry: No
Fred: Is there a lower and upper jaw?
Sherry: Yes
Fred: Is there any pigment lacking?
Sherry: No
Fred: Is the pattern abnormal?
Sherry: No
Fred: Is there a curvature of the spine?
Sherry: No
Fred: Is there an extension of the spine beyond the rump?
Sherry: No

Fred: Now we move on to the examination of the limbs. Are there four limbs?
Sherry: Yes
Fred: And two hind limbs?
Sherry: Yes
Fred: Is the hind limb complete?
Sherry: Yes
Fred: Is there a complete foot?
Sherry: Yes
Fred: Is there any skin webbing?
Sherry: No
Fred: Is there any evidence of a bone bridge? (This would be an abnormal shape of the hind limb)
Sherry: No
Fred: Any evidence of rotation?
Sherry: No
Fred: The digits on the hind limb – are there five digits?
Sherry: Yes
Fred: Is there complete fore limb?
Sherry: Yes
Fred: Is there any evidence of skin webbing?
Sherry: No
Fred: Any evidence of a bone bridge?
Sherry: No Fred: Rotation? Sherry: No
Fred: Are there all four digits?
Sherry: Yes, all four there.

Fred: The examination is complete.  We did not observe anything abnormal with this frog, and it 
would be released back into the pond.  If there was any abnormal appearance to this animal, we 
would put it in a small jar with holes and moist vegetation, take it back to the laboratory for 
photography and then decide whether it would be shipped for x-ray analysis.



Narrator: Release any tadpoles, adult frogs, and normal metamorphs back to the pond only after you 
have collected 50 metamorphs.  Hold the metamorphs in a cool, shady spot until you can release 
them. Set up your measurement station in the shade as well.

New Field Processing section shot in August 2004
Laura Eaton-Poole: Fred and Sherry showed us a way to process frogs in the field and that was a 
great way to do it when you’re first getting started and you’re not familiar with the types of 
abnormalities that you might run into, but you are processing fifteen to a hundred frogs per site, 
so it can be rather time consuming and laborious.  So once you are familiar with the abnormalities, 
you might want to use a little bit of a short cut that is still systematic.  What we often do is 
hold the frogs underneath the elbows so that their legs are dangling down, and then look at the 
body parts in the same order with each frog.  I look at the eyes making sure they are symmetrical 
and look normal.  Check the jaw.  Check down the back for any potential scoliosis. Check the front 
toes – four on the front.  Make sure they’re all the proper length, symmetrical on both sides.  
Dangle the hind limbs so that you can see that they are the same length and that he has five toes 
on each foot and that the toes are symmetrical on both sides.

We do have a few abnormal frogs that we can share with you.  These come from the state of Maryland. 
 I like to point out that it can be highly unpredictable when you’re going to run into abnormal 
frogs.  We can sample a site for a number of years and think it’s a reference pond, and then all of 
a sudden you’ve got abnormal frogs. We sample once in July – we have a limited season here in the 
northeast.  We can sample once in July and have all normal frogs and then sample again in August 
and get a crop of abnormalities and the types of abnormalities we see can be highly unpredictable. 
This guy is particularly interesting it’s actually a little amazing that he
is alive and probably wouldn’t be for long in the field.  I’ll show how we might process this 
fellow, trying to hold him underneath the elbow. You will note that we have no hind limbs what so 
ever.  This fellow has a really interesting abnormality, example of an eye abnormality.  As
you can see his right eye is enlarged and all black.  My guess is it’s an injury but we may never 
really know for sure exactly what has caused it. Here we have another abnormal frog from the same 
site.  He has a slightly shortened femur and below that the calf and foot are much reduced and 
disfigured.  One last thing we liked to mention is the importance of having clean hands when 
handling frogs.  They do have very absorbent skin, so if you have mosquito repellent on your hands, 
nicotine from smoking cigarettes, or hand lotion on your hands it could be a problem for the frogs. 
 So make sure that you clean your hands before you go out in to the swamp and get them muddy and 
dirty.  Now we’re going to head back into the lab to prepare these frogs for shipping.

Narrator: If you have arranged with a pathologist to examine live frogs, the following section 
demonstrates how to ship those study animals. All of the SOP’s are included in your manual. 
(19:38-20:05)

Packing & Shipping Live Amphibians
Laura Eaton-Poole: We’ve completed our field sampling, and since we did collect some abnormal 
frogs, we have decided to ship them for further analysis.  Possible destinations include Parasol 
otology, pathology, and radiography. It is really important that you contact the people

before you ship to make sure that they will be available to collect the frogs at their destination. 
It’s usually best to make sure the frogs arrive before Friday.  The types of containers you might 
want to use for shipping include hard plastic Tupperware containers, which is what we’ve used in 
the past.  You can use containers that you might have floating around the lab as long as you can 
perforate the lids.  We have found these new throw away Tupperware containers to work very, very 
well for us and it’s really easy to perforate the lids.  It’s also critical that you label each one 
of your containers with a frog number.  This is the same frog number that would correspond to the 
frog number that you put in the database.  As you put the frogs in the containers, give them their 
frog number, you also want to be filling out your shipping form that we’re going to be including in 
this shipment.  It’s very important that the frogs stay very moist during shipping.  One way of 
doing that is to use site water and site vegetation.  If you don’t have that available to you, you 
can use absorbent paper towels.  We recommend that you use paper towels that don’t have any 
coloring in them.  Some people feel strongly that you not use paper towels that have bleach in 
them; however, they can be hard to find and are not very absorbent.  We have been at times using 
the absorbent white paper towels, as far as I know they work all right.  It is very important the 
type of water you use.  You don’t want to use any water that has chlorine or salt in it.  So you 
can use well water or you can use drinking water that you got from the store.  So you want to 
moisten your paper towel.  Put it in the bottom of your container and get the frog in the container 
without mashing it with the lid. Once you have all of your frogs put into containers, size of the 
container does depend upon the size of the frog.  For instance, I have an adult frog in this 
container and a very small pickerel frog in this container. As you prepare to ship, you want to put 
cold blue ice on the bottom of your containers.  Cover the blue ice with flat newspaper.  It’s 
important that the frogs not have contact with the blue ice as it will freeze them and the idea is 
to get the frogs to their destination alive.  There’s a certain
amount of common sense here in that you want to have the frogs in there snuggled up.  I usually use 
crumpled newspaper to snug them up.  And then I put more crumpled newspaper on top just in case the 
cooler gets tipped over or tipped on its side.   Remember, whoever is shipping these frogs aren’t 
going to know you have live animals in there that they need to keep alive. Once you’ve completed 
your shipping form, we usually fold it up and put it in a zippy bag to keep it from getting wet.  
And tape it to the lid of the cooler.  And then you want to thoroughly tape down the cooler.  Wrap 
tape around it.  We label the coolers biological samples – we never write on them live frogs.  
Fedex usually doesn’t appreciate live animals in coolers, but you can check your protocols for more 
details.

Preserving Euthanized Frogs for Shipping
Narrator: You should euthanize suspected abnormal frogs according to the method recommended by your 
regional coordinator.  Not all survey teams will be required to conduct this step. This segment of 
the video guides you through the process of preserving euthanized animals.

Laura: You need a waxed bottom Tupperware container, ethanol, some surgical scissors, some pins, 
and surgical tape that I’ve already placed at the bottom of the table.
Now this container is essentially hand-made.  We melted beeswax on the bottom of the Tupperware. 
The advantage of this over a dissection tray is that you can place a top over it which is, since we 
will be pouring ethanol in it-there will be fumes coming out of it, so we highly recommend having a 
top.  If you can’t find beeswax like the Chesapeake Bay office found, the New England field office 
melted the wax rings you use at the base of a toilet.  I don’t think it worked quite as well as the 
beeswax but in a pinch it did work.  Now what’s most important for radiology is that the frog be 
flat, but since we are preserving in ethanol, it is also very important to open the body cavity 
when your preserving informal and it absorb into the body cavity readily, and preserves the organs, 
but ethanol isn’t absorbed as easily so we have to open the body cavity.  Then we flatten the frog 
out as best we can.  We try to spread the toes
since you can have abnormalities of the toes you want to try and get a clear x-ray of each of the
toes. This requires a certain amount of patience.  We are going to try and pin him flat.  Pinning 
into the flesh of the frog just doesn’t really work that well and the tape doesn’t really hold to 
the wax all that well.  So what we found we can do is tape the limb down and then pin the tape into 
the wax and that seems to work pretty well.  I’m going to do that right next to the limb, and it 
helps to have cut your tape in advance because it’s not that easy to work with, especially with 
gloves on.  Now, we are working with a normal frog for this demonstration. It’s recommended that 
you preserve the frog in 90% ethanol.  We just need to cover him.  You can leave him in here for a 
good two weeks and then move him to a jar with 70% ethanol -90% ethanol dries a little too much so 
use a more dilute solution for long-term storage.  Keep the top on it-keep the fumes down.

Narrator: Any chemicals that you have used in either your euthanasia or preservation procedure 
should be captured and disposed of properly.  Do not release any chemical into the environment. 
(29:50-30:03)

Decontamination
Sherry: Now we’re going to begin the decontamination process.  It’s important to do this between 
each sampling location or refuge and the reason we do this, this eliminates the spreading of 
pathogens from one site to another site.

Fred: So the first thing we do is use a hose, a garden hose, to spray off the mud and the big 
pieces of debris that are on either the net or the waders, and then we want to make about a 5% 
solution of bleach, which we will pour into a garbage can and we will fill the rest of the garbage 
can with water with the hose.  Then we just want the net and the boots to soak for at least 10-15 
minutes in this solution to really make sure that we’ve decontaminated any disease organisms.
So after the equipment has soaked for a while in the bleach solution, we use a brush, and we give 
it one final brushing to try and make sure we remove all the debris as best we can.  Now once we 
finished, we take our equipment out and we hose it down again with the hose so that the bleach 
solution is gone.  We don’t want to be carrying around bleach in our nets when we’re examining 
frogs. (30:28-32:47)

Sherry: If you have any further questions, please contact your Regional Amphibian Coordinator, the 
Washington Office Division of Environmental Quality, or Fred, Laura, or myself. (32:50-
33:00)