An Overview of the Histological Study of Marine Finfish

Histology is one way scientists at the Fish and Wildlife Research (FWRI) learn how fish function. This article describes the work done by the FWRI histology and finfish biology departments including techniques, methods and illustrative examples.

Histology is the science of producing stained sections of preserved tissue on glass slides that can be examined under a microscope. Parasites, bacteria, and fungi, as well as pathological processes and abnormalities can be detected in these preserved tissues. The techniques used in the Florida Fish and Wildlife Conservation Commission's (FWC) Fish and Wildlife Research Institute's (FWRI) histology laboratory are similar to those used in hospitals where medical doctors and pathologists examine tissue. Histology is an important research tool for numerous research projects at FWRI, including Fish Biology, Aquatic Health, Endangered and Threatened Species, and Shellfish Biology. The FWRI Histology Lab has processed tissue samples from dozens of plant and animal species (see list, PDF file, 16KB). Our tissue slides are used to better understand fisheries reproductive dynamics, the overall health of marine species that are important to Florida, and for the evaluation of pathologies and parasites.

The FWRI histology lab applied new techniques in the 1980s using plastic to embed fish tissue rather than the traditionally used paraffin. One major advantage of using plastic instead of paraffin is that the tissue can be cut thinner. Thinner tissue has greater clarity, lower distortion, and higher information content, thus enhancing our diagnostic capabilities. A disadvantage of embedding tissue in plastic is that it takes more time and costs more than embedding tissue in paraffin. These added costs are more than offset by higher quality results.

A crevalle jack (Caranx hippos) with muscle removed and stained muscle sections on slides as a final product.
Figure 1: A crevalle jack (Caranx hippos)
with muscle removed and stained muscle
sections on slides as a final product

At FWRI, small pieces of animal or plant tissue (less than 1 cm²) are first preserved in a fixative (preservative) solution (typically formalin). The tissue is embedded in a glycolmethacrylate resin (a type of hard plastic) and the plastic block with the tissue is then cut on a microtome. The microtome knife is made of glass, which cuts tissue at a thickness of 4 microns (less than 1/1000 of an inch). The thin tissue sections are mounted on a microscope slide and dyed with a variety of stains designed to highlight specific cell types or cell products such as glycogen or proteins.

 

A histological section of a tarpon ovary that can be used in finfish biology studies.
Figure 2
: A histological section of a
tarpon ovary that can be used in
finfish biology studies.
A histological section of fish muscle tissue with a fungal infection.
Figure 3
: A histological
section of fish muscle
tissue with a fungal infection.

Fixation/handling of samples for FWRI microscopic analysis

Proper handling and preparation of tissue specimens from the time of collection in the field to the time of staining in the histology lab is a critical step in obtaining good histological slides. Without proper preservation (fixation) the cells of living things undergo a process of autolysis, or autolyze ( see definition), beginning at the moment of death. This post-mortem degeneration can be confused with atresia, which is the living organism's process of resorbing cells. From the time specimens are received from the field, the following steps are followed by the FWRI histology lab to improve the chances for good results:

Specimen preparation:

1. Obtain needed materials/solutions before proceeding.

2. Prepare samples as follows:

IF LARGE (e.g., ripe fish gonads 6-10+ cm. in length and 4+ cm. in diameter or whole bivalves):

- harden in appropriate fixative (see #4) on ice for 1-2 hrs before slicing (see #3);
- submit a section no larger than 3 cm. in any dimension.

IF AVERAGE (e.g., 1-3 cm. diameter x 3-5 cm. length)

- submit entire gonad (preferably both lobes joined intact),
or
- select a portion of the gonad and slice it (see #3) no smaller than 1 x 1 x 1 cm.

IF SMALL (e.g., "stringy," thread-like, or less than 1 cm. in longest
dimension)

- submit entire gonad intact.

3. To slice a sample prior to fixation, use a sharp, single-edge razor blade
and a smooth slashing motion. Do not use a scalpel (will not cut straight)
or a back-&-forth sawing action (tears up tissue).

Tissue fixation: (Fix on ice or in the refrigerator, as lower temperatures retard autolysis during penetration of the fixative.)

4. Fix the samples immediately in one of the following:

- Standard FWRI fixative is 5% PFMA (Paraformaldehyde) in 0.1M Phosphate Buffer, pH 7.4
- 10% formalin (prepared from 37% Formaldehyde) in 0.1M Phosphate Buffer, pH 7.4
- 10% Neutral Buffered Formalin purchased commercially
- Other fixative as agreed upon

If immediate fixation is not possible, pack the samples thoroughly and completely ON ICE to retard autolysis. Make certain that the tissues are NOT in direct contact with the ice!

Ratio of fixative to tissue: 10:1 v/v (volume of fixative to tissue volume) is a MUST MINIMUM... 20:1 v/v is OPTIMUM.

Use a sample jar large enough to accommodate the volume of fixative required for the size of the sample!

5. Duration of fixation:

- large samples (ripe gonads or bivalves) = 3-5 days;
- average gonad samples = 24-48 hours;
- biopsies = 4-6 hrs.

6. After fixation, rinse each sample with four 15 minute tap water changes.
For large samples, the rinsing time should be extended to four 30 minute
tap water changes. Place the samples on a rotator if possible or gently swirl
the samples in the rinse water periodically. The purpose of the water rinses
is to remove excess fixative and buffer salts from the tissue. The buffer salts
(and sea water) are generally incompatible with subsequent ethanol
treatments, and insoluble crystals or precipitates may form within the tissue,
preventing or hindering histological sectioning of paraffin or glycol
methacrylate blocks.

7. Replace the final tap water rinse with 70% Ethanol (EtOH). The FWRI Histology Lab uses recycled 70% ethanol which has been tinted pink to distinguish it from other solutions (such as formalin and to prevent the re-use of recycled 70% EtOH) for economic savings. If recycled EtOH is not available, use 70% Ethanol prepared by diluting 95% Ethanol with distilled water.

The Stains

When we stain our clothes, we usually rush to do whatever is needed to remove the stain. With tissue samples the histology lab does just the opposite. Histologists do everything possible to ensure stains infiltrate every nook and cranny of a sample. Why do we stain the tissue samples? Because different stains and combinations of stains have been proven to color structures within individual cells, or microstructures, in predictable ways (Table 1). For example, the stain known as hematoxylin-eosin (H&E) colors the nuclei of cells a deep violet-blue whereas cytoplasm will be stained varying shades of pink and orange. This is because hematoxylin binds to acidic components of the tissue and eosin binds to the basic components of a tissue. This is helpful to scientists who need to identify particular tissue types or developmental stages. The stains most commonly used at FWRI are named below. Select a stain to view a PDF explaining its use and effects in greater detail.

Table 1 - Different stains color cellular microstructures in different ways. Select a stain to view a PDF file explaining its use and effects.

Stain name

Cellular
structure

H & E
(Hematoxylin- Eosin)


PAS/MY
(Periodic acid -
Schiff's reagent - Metalin yellow)

Thionin

Nucleus (non-ovarian) Deep blue-violet Deep blue-violet Deep blue with distinct chromatin
Nucleolus (non-ovarian) Orange-pink to
red-violet depending
on cell type
Blue-grey to
yellow-grey

XXX

Chromatin (non-ovarian) Blue-black Blue-black Deep blue
Cytoplasm (non-ovarian) Varying shades of
pink and orange
Shades of yellow
or yellow-tan
Light blue
Collagen Pale pink Magenta

XXX

Muscle Deep pink Bright yellow

XXX

Nuclei of
primary oocytes

Deep blue-violet

Pale yellow-grey

XXX

Chromatin of
primary oocytes

Blue-black

Pale yellow-grey

XXX

Cytoplasm of
pre-vitellogenic oocytes

Varying shades of
pink and orange

Deep blue-violet

XXX

Erythrocytes
(red blood cells)

XXX

XXX

Green
Mucus

XXX

XXX

Red-purple to violet
Cartilage matrix

XXX

XXX

Red-purple to violet
Mast cell granules

XXX

XXX

Deep violet

Histology applied

One important way histology is commonly used in fisheries is the study of fish reproductive tissue. Although macroscopic evaluation of gonads can provide important information, the ability to examine reproductive tissue at the microscopic, or cellular, level gives biologists a powerful tool to understand the details of fish reproduction. Histology of ovarian tissue is commonly used to better understand: (1) size/age at maturity; (2) daily and seasonal pattern of spawning; (3) spawning location; and, (4) fecundity (see definition).

Histological analysis is often used to determine whether the pattern of oogenesis, or egg growth is asynchronous (see definition) or synchronous (see definition). Determining this is critical because it will indicate both the spawning pattern of a fish (i.e., total spawners or batch spawners (see definitions) and what types of methods are necessary to estimate annual fecundity. With batch spawning fishes there is an additional need to determine whether they have determinate fecundity (see definition) or indeterminate fecundity (see definition). For batch spawners with indeterminate fecundity it is necessary to determine both batch fecundity (number of eggs spawned at any one time) and spawning frequency (or how many times any one fish will spawn in a season).

As the use of histology to evaluate reproductive condition in fishes has grown, so too have the number of histological classification schemes and the terminology used with them. Although there are several terms which are frequently used, the same term can often mean different things to different scientists. In addition, developmental stages are often referred to by number, but these numbers signify different classes depending on the classification scheme being used. In an effort to allow better communication and comparison of different species' reproductive strategies, a group of scientists from the United States and Europe have been working together to develop a conceptual model of the reproductive cycle which can be applied to all fish species. This conceptual model was presented as a poster at the American Society of Ichthyologists and Herpetologists (ASIH) and American Fisheries Society (AFS) 2007 national conventions. The working group is currently developing a manuscript. Because of the interest at these meetings, we are posting here both the abstract (PDF file, 1 KB) from the poster and the handout (PDF file, 1 KB) that was available outlining the phases in the model and their criteria.

Histological analysis of many fish over time paired with other fisheries information such as time and place of capture, age, and size of the fish allows fishery managers to have a measure of the reproductive health of a stock and to develop management strategies to protect spawning populations.

Table 2 - Histological techniques allow side-by-side comparison of cellular micro-structures of fish from different species at the same stage of development. Select a thumbnail for a larger view of the photo (requires Adobe Acrobat Reader).

Histological
criteria

Spotted seatrout
(Cynoscion
nebulosus
)

Mutton snapper
(Lutjanus analis)

Yellowtail snapper
(Ocyuris chrysurus)

Primary growth - Immature

Spotted seatrout - Primary growth immature phase

Mutton snapper - Primary growth mature phase.

Yellowtail snapper - Primary growth immature phase.

Primary growth - Mature

Spotted seatrout - Primary growth mature phase.

Mutton snapper - Primary growth mature phase.

Yellowtail snapper - Primary growth mature phase.

Cortical alveoli

Spotted seatrout - Cortical alveoli phase.

Mutton snapper - Cortical alveoli phase.

Yellowtail snapper - Cortical alveoli phase.

Vitellogenesis

Spotted seatrout - Vitellogenesis phase.

Mutton snapper - Vitellogenesis phase.

Yellowtail snapper - Vitellogenesis phase.

Final oocyte maturation

Spotted seatrout - Final oocyte maturation phase.

Mutton snapper - Final oocyte maturation phase.

Yellowtail snapper - Final oocyte maturation phase.

Ovulatory


See Figure 4

Post-ovulatory

The post-ovulatory stage in a spotted seatrout.

No image available for this fish and phase.

No image available for this fish and phase.

Atretic

Spotted seatrout - Atretic phase

Mutton snapper-Atretic phase

Yellowtail snapper - Atretic stage

 

20070725_223522_22678.jpg

Figure 4 - The ovulatory stage in a spotted seatrout. Because this stage is so short in duration, it is rarely observed and photographed histologically. We are pursuing photographs of this stage for the other species. ( return to table)


Glossary

Autolysis - Autolysis means the destruction of a cell after its death by the action of its own enzymes, which break down its structural molecules. With tissue samples intended for histological study, this process must be delayed with the use of icing, freezing and/or chemical preservation.
Fecundity - The total reproductive output of an organism or population. In fish, this is expressed as total number of eggs in a spawning season.
Fecundity, determinate - The number of eggs is fixed because all eggs are produced prior to the spawning season. Total egg production can be estimated by counting the number of developed oocytes in an ovary prior to the spawning season. This is demonstrated when oocyte size is plotted on a graph and a distinct gap is seen between the most developed and least developed oocytes.
Fecundity, indeterminate - The number of eggs is not fixed prior to the spawning season because eggs continue to be produced to replace those that have been spawned throughout the spawning season.
Oocyte - A female germ cell in the process of development. It is the precursor of an egg.
Oogenesis - The formation, development, and maturation of an ovum. The formation, development and maturation of oocytes.
Oogenesis, asynchronous - Oocytes are continually developed.
Oogenesis, synchronous - All oocytes in an ovary are developed on the same schedule.
Batch spawner - A fish species that releases eggs multiple times throughout the spawning season.
Total spawner - A fish species that releases eggs only one time per spawning season.



FWC Facts:
More than 1,000 different species of fish populate Florida's inshore waters.

Learn More at AskFWC