An Overview of the Histological Study of Marine Finfish

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). 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.

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, 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.

• H & E (Hematoxylin- Eosin)
• PAS/MY (Periodic acid -Schiff's reagent - Metalin yellow)
• Thionin

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.

Histological analysis is often used to determine whether the pattern of oogenesis, or egg growth is asynchronous or synchronous. Determining this is critical because it will indicate both the spawning pattern of a fish (i.e., total spawners or batch spawners 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 or indeterminate fecundity. 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 from the poster and the handout 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. 

See Figure 4