Troubleshooting Photomicrography Errors

Microscopy Primer
Color Negative Film Processing Errors
Light and Color
Microscope Basics
All color films consist of at least three black & white emulsions, coated one on top of the
Special Techniques other to form a permanent, multi-layered structure supported by the film base. The top
Digital Imaging emulsion responds only to the blue third of the spectrum, while the middle and lower
emulsion layers respond only to the green and red thirds, respectively. Each layer of a color
Confocal Microscopy negative film emulsion absorbs a specific subset of the entire spectrum of visible light
Live-Cell Imaging wavelengths, prescribed by the properties of the dyes embedded in that layer.
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Other colors will react with two or more of the emulsion layers to reproduce that color when
Site Info the negative is printed. Color negative film produces an image that is complementary with
Contact Us respect to colors and density of the original microscopic viewfield. Subsequently, the
Publications negative is printed, usually onto color photographic paper, to yield a positive color print.
When negative film is processed, the developer converts light-sensitive crystallites of silver
Home halide (usually the bromide salt) compounds in the emulsion layers into metallic silver. While
doing this, the developer also oxidizes and combines with dye couplers that are either built
The Galleries: into the emulsion layers or added during development. The result is the formation of three
dye layers, one from each of the subtractive primary colors: cyan, magenta, and yellow. The
Photo Gallery blue-sensitive layer of the original film forma a yellow image, while the green and red-
sensitive layers form magenta and cyan images, respectively. Bleach is then utilized to
Silicon Zoo remove all of the silver metal, and each layer is left with only a color image. Color negative
Pharmaceuticals film has an orange mask that is used to help control contrast and correct for deficiencies in
green and red-sensitive layers when the negatives are printed.
Chip Shots
Phytochemicals The specimen employed in this discussion is a brightfield color photomicrograph of a
DNA Gallery quadruple-stained thin section of longitudinal tracheids derived from loblolly pine. The
staining mixture consists of safranin O (nuclei, chromosomes and cell walls), fast green
Microscapes (cytoplasm and cellulose cell walls), crystal violet (starch), and orange G (acidophilic
Vitamins cytoplasm). A Kodak Wratten CC10M magenta color compensating filter was used in the
light path to eliminate a green cast present in background of the stained specimen. The
Amino Acids
image was recorded on Fujicolor Superia 100 color negative film and processed using the
Birthstones Kodak C-41 technique at 37.8 degrees centigrade.
Religion Collection
Processing color film requires considerably more effort and attention than does black &
Pesticides white film. Although the basic steps are similar in nature, color processes require more
BeerShots steps and greater temperature control than do black & white processes. The stability of
chemical solutions is also a factor in color processing, because sensitive combinations must
Cocktail Collection
Screen Savers be mixed immediately prior to use to avoid rapid deterioration of the components. In
general, the shelf life of diluted color process chemicals is far less than those used for black
Win Wallpaper & white film processing.
Mac Wallpaper
When color negative film is processed, the first step (color developer) is designed to initiate
Movie Gallery
reduction of silver halide salts in each of the three emulsion layers into metallic silver that
produces a negative image. This step is common to all color photographic materials
regardless of whether they ultimately produce positive or negative images on film or paper.
After the silver has been formed, exhausted agents reach with dye couplers to
simultaneously form colored dye images, which represent the complementary color to the
wavelengths of light that were originally recorded by the film. This process is commonly
referred to as chromogenic development. In the emulsion, yellow dye is formed and
coupled to the blue-sensitive layer, magenta dye is formed in the green-sensitive layer, and
cyan dye is formed in the red-sensitive layer of the film.

The next step is a bleach solution, which actually stops the development process and
oxidizes black silver metal back into silver halides that can be fixed out together with other
unprecessed halides. Fixer is either used alone (Kodak C-41 process), or coupled with a
fixer in the same solution (called a bleach/fix), in "hobby" kits that feature truncated color
negative process solutions. After bleaching and fixing, the film is washed to remove fixer
and other soluble by-products, then rinsed in stabilizer to improve dye stability and harden
the emulsion.

Most brands of color negative film are processed by the Kodak C-41 color processing
system of chemicals. Similar processing chemistry is also offered by aftermarket
manufacturers such as Beseler, Unicolor, Photocolor, and Tetenal. The processing steps are
identical for all C-41 type processes and include a color developer, bleach, fixer, bleach/fix,
final wash, stabilizer, and drying. Provided temperature control can be established (a
constant 37.8 degrees centigrade +/- 0.1 degree), this process can be conducted in the
laboratory using small daylight developing tanks, or if the expense account allows, more
complex motorized rotary-tube film processors with built-in water baths and temperature
control. Commercial roller-drum tank units for processing color negative film are available
for under $1000. Truncated C-41 process kits that simplify the process by combining
several of the steps are also available for amateur photographers. These simplified
processes contain a color developer, fixer, combined bleach and fixer, and a stabilizer bath.
Although they also require strict control of temperature, the aftermarket "hobby" C-41 kits
are much easier to use and do have somewhat more temperature latitude than do standard
C-41 processes.

The color developer is the critical part of the C-41 (or hobby kit) process, and it is essential
that the temperature be held to within the specified limits. The bleach, fixer, washes, and
stabilizer should also be maintained under tight temperature control, ideally at the same
temperature as the color developer, although the specified range is usually 24 to 41
degrees Centigrade. C-41 hobby kits by Kodak and other manufacturers can be used to
develop 16 rolls of film with 24 exposures in the magazines. As the solutions age, extended
processing times are necessary to achieve the desired effects. Details on the time
increases are given in an instruction booklet supplied with the kit. It is probably better to add
one to two minutes to the color developer time given and about five minutes to the
bleach/fixer time to insure adequate bleaching of unwanted dyes.

Because the C-41 process color developer (the first step) has such a short process time
(three and a quarter minutes), agitation is extremely critical. The pre-warmed solution
should be rapidly added to the development tank and agitation started immediately. After
the color developer has completed its alotted time, it is discarded and the film is rinsed
thoroughly with wash water. The last starges are typically a pre-bleach solution, designed to
stop chemical carry over, followed by bleaching, fixing, and washing exhaustively to remove
all solubilized silver salts.

Development is the critical stage in processing color negative film. Most films of this type
will not tolerate development changes because color is affected along with film density and
contrast. Some color negative films are designed to allow adjustment of color development
times (push or pull processing) to compensate for exposures made either at lower or
higher ISO ratings than the film emulsion is registered for. When overdeveloped, most color
negative films demonstrate color casts that are different in shadowed and highlight regions.
In addition, pushed film has a high level of fog and grain.

Examination of processed color negative film is an important

step in evaluating the results of photomicrography experiments.
The first step is to carefully check the film for proper exposure
and errors that may have been introduced by color balance,
heat reducing, neutral density, and compensating filters. Color
negatives that are very dense and dark have been
overexposed, a condition that occurs when too much light
reaches the film plane. This is not a film processing error, but
rather demonstrates incorrect use of the microscope. Another
common photomicrography error with color negative film is
underexposure, where the film density is very thin because not
enough light has reached the film. These and other color negative film errors are thoroughly
discussed in our section entitled Troubleshooting Color Negative Film Errors, which
describes common errors encountered in photomicrography using this type of film.

Evaluation of color negatives is somewhat difficult and requires more skill and experience
than do black & white negatives or color transparencies. Highlight areas should not be
excessively dense and shadowed features should be sufficiently detailed to render an
overall balanced photomicrograph. Judging specific colors in color negatives is complicated
by the presence of a mask that produces an orange tint over both the sprocket holes and
the image area, including the color dye layers. The purpose of this mask is to help control
contrast and correct for deficiencies in green and red-sensitive layers when the negatives
are printed.

In many instances it is difficult to distinguish between errors that occur during processing
and those that arise as a result of mishandling the microscope. Some errors are extremely
subtle in nature, such as small contrast discrepancies and unusual density shifts, but others
are often blatant. A variety of the most common errors encountered in color negative film
processing are discussed in the paragraphs below with accompanying illustrations derived
from photomicrographs of the specimen described above.

Overdevelopment with color negative films in the C-41 process

leads to very dark contrasty negatives that have dense
highlights lacking in detail with a dramatically increased grain
size. In many instances, edge numbers and letters often appear
very dark, too. This error can occur when film is left in the color
developer for more than the recommended time, even when
specifically intended during push-processing (which, by its
nature increases grain size). The same effect will occur if the
temperature is too high or the agitation is too vigorous. Carefully
monitor the water bath temperature during the C-41 process,
especially when using the color developer solution. The
temperature should remain at 37.8 degrees Centigrade without any significant deviation to
avoid process errors. Other bath temperatures (bleach and fixer) are not as critical and can
afford a few degrees of fluctuation.

Underdevelopment when processing color negative film using

the C-41 method leads photomicrographs that are low in
contrast, although the image does contain some detail in
highlight regions and midtones. Typically, when the color
developer time is cut too short, resulting negatives appear
obviously underdeveloped and are very thin. Another cause for
underdevelopment with color negative film occurs when the
water bath temperature falls below the recommended level of
37.8 degrees Centigrade. The C-41 color developer step is so
sensitive to temperature that even if the drop is only 0.5
degrees, there will be a noticeable decrease in film density. It is
a good idea to pre-heat both the color developer (and other processing solutions) as well as
the developing tank for at least 15 minutes prior to initiating film processing. Similar results
are obtained when the film is insufficiently agitated during the development process. The
color developer must be poured into the developing tank very quickly and agitation must
start immediately. Underdeveloped film should be carefully examined on a light table (5500
K) to determine if it is salvageable. There is a possibility that underdeveloped film can be
restored, at least to a certain degree, with chemical dye reducer.

When every frame in a color negative filmstrip has been only

partially developed, then one edge of the entire roll will appear
much light than the opposite edge. This occurs when there is
not enough color developer solution in the processing tank to
completely immerse the film. To avoid the problem in the future,
carefully measure the volume of liquid necessary to cover all
film reels inside the developing tank and use this measurement
to adjust developer solution volumes in subsequent processing
sessions. Roller drums that are not properly filled will produce
partially developed film frames positioned at only one end of the
strip, nearer the center of the film reel. Film that has been only
partially developed is permanently damaged and will not produce good prints unless
enough of the image remains to allow satisfactory elimination of undesirable areas by
cropping.

Unusual or unrealistic color casts or an obvious lack of color

balance is often the result of a contaminated color developer
solution. The source of contamination could range from unclean
processing equipment (developing tank, graduated cylinders,
mixing bottles, etc.) to mistakes mixing the processing
chemicals. It is a good idea to use the same mixing bottles for
each solution, even through they are washed out at the end of
the processing session. By carefully labeling each bottle,
mistakes are kept to a minimum. First developer solutions that
have been diluted to working volume, then left for lengthy
periods of time before use, can also produce strange color
shifts in addition to underdevelopment. Even when the process
temperature is raised to compensate for aged chemicals, reactions in the film emulsion
during development with old solutions often leads to disastrous results.

Clear film with no edge marking numbers or letters is usually

the result of omission of the bleach/fix stage, but could also
result if the color development is forgotten. This error is easy to
differentiate between unexposed film, which is also clear, but
will display black or red edge numbers. If the film has been
processed successfully through the color developer, there
should be some detail present when examined through a bright
light, such as a fiber optics illuminator. Hold the negative up to
the illuminator, and examine the surface with a 10x loupe. Some
detail should be visible (although very faint), which may indicate
that the film can be salvaged by processing in the bleach/fix
solution. If extra solution is available, there is nothing to lose by attempting to properly
bleach the dyes and fix the film, even if it has gone through a wash and drying cycle.
However, if the film was placed into an aldehydic stabilizer bath prior to drying, then it is
pointless to try and bleach unwanted dyes, which are now chemically fastened to the
emulsion. There is no point in trying to recover the film if the color development stage is
omitted.

When the film is totally clear, but edge markings are present
and have been properly developed, there is a possibility that the
film has not been exposed. This can be indicative of a serious
problem with the film advance mechanism on automatic
cameras or a malfunction of the camera shutter system. If the
entire roll is unexposed, yet the camera appeared to be
functioning properly during photomicrography, check to make
certain the camera is receiving light from the microscope. Also
examine the take-up spool to make sure it is working properly
and the film is correctly attached. Many automatic exposure
systems will not allow film to be exposed when insufficient light
is being received, but older manual systems do not have this safety feature. In film rolls
where only a single or couple of frames are unexposed, check the camera to make sure film
is advancing properly.

If color negative film is inadequately bleached, the result is less

than normal amounts of cyan dye is formed because some of
the dye remains in the leuco (colorless) condition. Often, film
that has not received enough time in the bleach solution will
have an overall red cast or small red spots (from lack of cyan
dye) on the emulsion. Another indication of inadequate
bleaching is the presence of a grayish-brown fog on highlights
with corresponding shadow detail that is lacking or too dark.
This is due to retained silver salts that have not been removed
by the bleaching chemicals, either because of insufficient
agitation, process time, or a depleted solution. In many cases,
bleaching errors can be corrected by rolling the film back onto reels and re-bleaching the
entire roll. This remedy will only work if the film has not been subjected to fixing in the
stabilizer bath.

If the film is fixed before bleaching or if bleach/fix solution

contaminates the color developer, then color balance, tonal
qualities, and overall film density will be compromised.
Contamination of the color developer with bleach or fixer will
render color tones a bluish color and may reduce or reduce film
density, depending upon the concentration of the contaminant.
In fact, any contamination of a developer with another solution
usually results in a color shift and reduced color density.
Subjecting the film to fixing prior to the bleach step prevents
removal of the silver complexes normally formed during
bleaching. In this case, film can often be recovered by
bleaching for the recommended period (or even extending bleach processing time by 25
percent), followed by fixing, then washing and stabilization. Small amounts of bleach/fixer in
the color developer solution will also result in bluish color tones that are impossible to cure.

Inadequate fixing usually results with incomplete removal of all

the sensitizing dyes and silver halide in color negative film
emulsions. A consequence of this is a shift of the emulsion
towards red and green color casts and an undesirable milky or
cloudy appearance of the negatives. This error occurs when the
fixer has been diluted by excessive solution carryover (in large
processes), inadequate fixing time, underreplenishment, and
fixer sulfurization. The process temperature has very little effect
on fixing rate if all other fixer solution conditions are within
tolerances. In some instances, a lack of agitation may cause
incomplete fixing of the negatives. Color negatives that suffer
from inadequate fixing are easily remedied by reprocessing in the fixing solution for the
appropriate time.

Dark or light crescent-shaped marks (or "half-moons") can

appear anywhere on the negative, including near the take-up
spool sprocket perforations, and are due to mechanical
wrinkling or creasing of the film. This error usually occurs during
loading of film into the tank prior to processing or during
removal of processed film from tank reels. The size of the mark
is an indication of the severity of damage to the film base,
especially when some of the emulsion has been removed. If the
damaged area does not cover the entire negative, it may still be
possible to get a usable print by cropping to include only good
portions of the film during printing.

Water marks are light or dark gray spots that occur when water containing a significant
amount of dissolved salts (hard water) dries on the emulsion surface. Careful use of hypo
clearing agent or similar wetting agents followed by squeegeeing the film will help alleviate
this problem, but will not guarantee a cure. Spots having a dark outline are caused by water
coming into contact with the film surface after the emulsion has dried. This mistake usually
occurs during cutting and packaging of the film and can be avoided by careful handling of
 film after processing and drying. Water marks can often be
removed with film cleaner without compromising the integrity of
the emulsion surface.

Air bells are circular light spots that

form in regions where the film
developer was unable to reach the
emulsion as a result of air bubbles
clinging to the surface. The easiest
way to avoid air bells is to gently
tap the developing tank on the
laboratory benchtop just after the
developing solution has been
added. In addition, the developing
solution should be agitated
according to the manufacturer's instructions during processing to dislodge any air bubbles
that subsequently form on the film. On some occasions, air bells appear reddish or blue
when they occur during fixing.

Torn sprocket holes can be an indication of a serious problem

with the camera or darkroom procedures. Generally, damage to
sprocket holes (the perforations used by the takeup spool to
advance the film) occurs because of a problem in the camera
advance mechanism. Frequently, the problem is caused by
rewinding the film by force without first engaging the rewind
mechanism (a button on the camera body). Another common
cause of torn sprockets is trying to forcibly advance film after
the end of the roll is reached. In many instances, the film is torn
off the spool, compounding the problem. When the film has
been mishandled during loading or unloading of the developing
tank, damage to sprocket holes can also occur. This is because film is usually loaded into
spiral grooves in a plastic or metal spool before being placed in the development tank. If the
film is forced into the grooves or rapidly extracted, some damage can occur if one of the
sprocket holes becomes caught in the reel or spool. To investigate this type of damage,
carefully examine the camera back to determine if the problem lies with the takeup reel or
some other portion of the film advance mechanism. If not, then review darkroom technique
to see how it can be improved.

The presence of contaminating fingerprints on the emulsion will

be obvious and is usually destructive to the film. Touching dry
film with moist fingers is the usual source of this problem, but it
can also occur during processing when the wet and soft
emulsion surface is accidentally handled. If the fingerprints are
due to developer solution, then resulting marks will be light and
usually impossible to remove. When the marks are caused by
touching the film with fingers that are wet from fixer chemicals,
then darker colored fingerprints will be left on the emulsion that
can sometimes be removed by thorough washing. To avoid
fingerprints on film surfaces, rinse and dry hands after they
have been exposed to chemical solutions and handle film by the edges, not the surface.
Fingerprints due to grease and oils can sometimes be removed by rewashing film and
treating it again with a wetting agent, or by cleaning the film with a cotton swab or film
cleaner. Handling film with cotton editing gloves will avoid fingerprint problems.

Long thin scratches that run parallel to the length of a filmstrip are caused by tears and
partial removal of the emulsion by mechanical force. This usually occurs during
squeegeeing, but can also occur in the camera back or during processing. Check the film
pressure plate on the camera back for damage or raised areas that might be scratching the
film as it advances or rewinds. If the film has been forcefully loaded into the developing
spool, sometimes the emulsion is rubbed too hard against the spool, causing blemishes.
Film bulk loaders are often the source of scratched film, but this is usually not obvious until
the film has been processed. If the error occurs repeatedly, then carefully examine film
loading and darkroom technique to uncover the source of the problem. Film that has been
damaged in this manner cannot generally be repaired, but one remedy (for printing and
 scanning purposes) is to gently rub some skin oils (from the
forehead or nose) into the scratches for a quick fix. Eventually
the oil will collect dust and should removed with film cleaner
before storing the transparencies.

Large blank spots or patches on the

emulsion surface are usually either
opaque or very dark in shade when
viewed on color negative film.
Occasionally, blank patches will
appear clear on the negatives,
creating undesirable dark spots on
resulting prints. The usual cause is
two loops of film that have stuck
together in the spool or reel during
development. Often, to conserve
space in developing tanks, photographers will stack several filmstrips together in the same
reel, or will inadvertently wind two loops together on stainless steel reels.

Squeezing reels too tightly in the developing tank can also

cause film to overlap and touch. When this occurs, developing
solution is prevented from reaching the film emulsion at the
point of contact, and the film will not be adequately processed.
In most instances, the affected area will remain totally
unprocessed and opaque, but can become clear if the film
separates later in the development process. To avoid the
problem, check to ensure film is winding correctly on the reel,
and listen for crackling or other unusual sounds when loading
film reels in the darkroom. If the winding doesn't proceed
smoothly, unwind the film and start over again.

Repetitive streaks originating from the sprocket holes and

traveling down into the emulsion are caused by too much
agitation of the developer solution. The primary goal of agitating
the first developer solution is to ensure an even spread across
the film surface and to continuously replenish depleted
chemicals in the area immediately adjacent to the emulsion.
Excessive agitation can force too much fresh developer into
contact with the emulsion, causing an overdevelopment
problem. The solution to this processing error is gentle, yet
sufficient agitation of the film according to the manufacturer's
specifications during processing. Overdeveloped film is often
damaged beyond repair and should usually be discarded.
Exceptions do occur however, and film that is not too
overdeveloped can sometimes be printed satisfactorily.

Uneven development is manifested by light patches and streaks

on the color negative film and is caused by insufficient agitation
of the film developing solution. When the agitation level is not
high enough, depleted chemicals near the emulsion surface are
not replenished in time to complete the development process
and can accumulate and slide across the film. This retards film
development in large areas causing streaks that compromise
the integrity of the negatives. Although film that is streaked
cannot be repaired, future development sessions can be
successful if the manufacturer's recommended agitation rates
are followed. Negatives that have been streaked by uneven
development usually yield unsatisfactory prints, but if the affected area is small, then
judicious cropping can often produce a reasonable print.

Cloudy film, a milky texture that occurs across the entire emulsion surface, is probably due
to inadequate bleach/fixing or contamination of mixing vessels or processing equipment.
This error is also common when one of the developer solutions is contaminated with
another solution. When the film is first removed from the developing tank, it should be
 carefully examined for processing errors. If the film appears
cloudy, immediately place the film back into the development
tank and load another batch of fixer or bleach/fix solution.
Agitate for approximately five to ten minutes and reexamine the
film. If it still appears cloudy, then the fixer is depleted or too old
and should be replaced. There is plenty of time to prepare a
fresh batch of fixer while the film is kept immersed in water. On
some occasions, film that appears fogged or cloudy may have
been permanently damaged by light leaks, heat exposure, or
irradiation by X-rays at airport checkpoints. Laboratories that
use radioactive chemicals capable of gamma radiation should
also store and use film in locations far removed from radioactive chemical work areas.

Reticulation occurs when film is exposed to extreme variations

in processing temperature. The negative image appears
"crinkled" or cracked throughout the surface and is damaged
beyond repair. This error usually occurs when heated developer
is inadvertently placed into the developing tank immediately
after mixing. If the color developer bath is too warm, reticulation
can occur to a lesser degree. As the degree of reticulation is
reduced (by using cooler solutions), the cracked surface is no
longer a problem, but film grain size often becomes increased.
The solution to this problem is to carefully monitor the
temperature of all solutions used in the development process.
On rare occasions, reticulation can lead to production of prints with some degree of artistic
merit, but most film damaged in this manner yields unsatisfactory prints.

Fogging is the result of a light leak, either in the film

development tank, darkroom (light leaking through the door, an
inadequately blocked window, or a safelight), or in the camera
back. Usually several frames are destroyed by fogging, but not
the entire roll. Check all potential problem areas to ensure the
mistake is not repeated with the next roll. Light leaks in the
development tank can occur because the lid is not properly
secured, or because the light-tight solution tube that runs
through the center of the film reels is not seated properly. In
rare cases, development tanks are cracked when they are
slammed against the counter to dislodge air bubbles. Darkroom
light leaks can be detected by turning off the lights and waiting for 15 minutes, allowing the
eyes to become accustomed to the darkness before checking around door and window
edges.

Static marks occur when film is unrolled too quickly in a

darkroom under conditions of extreme low humidity, usually
during the winter season. They appear as scratches, spots, or
lightning-like black lines caused by static electricity that is
prevalent in dry conditions. Static marks can also be caused
inside the camera back or in bulk loaders when film is advanced
or rewound too rapidly in dry weather. To avoid this problem,
always take precautions when loading or unloading cameras
and developing tanks when the relative humidity drops below 35
percent, especially in high pressure areas. Film, whether
processed or unprocessed, should always be handled carefully
using cloth gloves to avoid static discharge and other forms of mechanical and chemical
damage.

Grit and dirt on the negative surface is usually caused by inadequate washing or improper
use of wetting agents. Make certain that wash water circulates well and is replenished often,
and that wetting agents are properly diluted (they can leave a residue when used too
concentrated). Damp film readily attracts dust and dirt, so thoroughly dry film before
handling. Areas that have extremely hard water are prone to this type of problem. Filtering
water before using it for photography processing is always a good idea, but in areas with
hard water, it can become critical. Mix commercial wetting agents with distilled water for the
best results.
 Processing color negative film in the laboratory has a number of
advantages and may be less expensive than using commercial
processors in the long run. It is definitely faster and more
convenient to process film at a local drug store or supermarket,
but if quality control and special techniques are important, then
it is probably better to process in-house. Some popular and
redily available color negative films that are easily developed in
the laboratory are Agfacolor, Fujicolor, Kodak Vericolor, and
Gold Plus films.
Contributing Authors

Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive.,

Melville, New York, 11747.

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State
University, Tallahassee, Florida, 32310.

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