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Volume IV - 4.4 Basic Analysis

DFS Pyramid Logo

Orientation and Training

Food and Drug Administration

DOCUMENT NO.:

IV-04
VERSION NO.: 1.5

Section 4 - Microanalytical and Filth Analysis

EFFECTIVE DATE: 10/01/2003REVISED: 02/14/2013

 4.4.1 Sample Analysis

A. Sample Analysis

  1. Guidance

    Before beginning each analysis, the analyst should ask their supervisor if any administrative guidance has been issued concerning the product about to be analyzed. Analysts should be aware and familiar with the guidance or program for which the product was collected under, shown on the Collection Report as the "PAC/PAF" number.  The analyst may also want to study and gain insight into the history of the product or the firm, pest problems, processing, and production as it relates to the agency's regulatory policy and the philosophical/economic impact on the regulated industry.
     
    Sources of information range from the Sample Collection Report (C/R, OASIS and/or FACTS), the supervisor and senior analysts, the sample collector, the EIRs in the firm's jacket, the FD484, and photographs taken by the Investigator. The analyst can request additional information not commonly found with the sample. 
     
    While reading the documentation provided with the sample, the analyst asks the following questions: (who, what, when, where) 
    • What does the analyst need to determine (problem area) for this sample? (i.e. insect, rodent, mold, and/or particulates- like glass or metal adulteration). 
    • What is the scale of the analysis? (i.e. visual, macro and/or microscopic?)
    • What methods, preferably official, are found for this kind of analysis?
    • What equipment and reagents will be needed, and how much of each is needed?
    • What steps/timeline will be followed before, during, and after the analysis?
    • Is there enough material/sample/time to do the analysis? E.g., can the analysis be modified such that one can accommodate the situation?
    • Does this analysis require a 702(b) portion and is there one provided?
    • Will help be needed, and when will it be needed? 
    • What kind of problems/interferences can one expect to see, and what can be done to avoid them? E.g., does the ingredient label show any unexpected, unusual ingredients that the analyst will have to deal with by method modification?
    • Is there any additional analysis needed, if so, what, when, where, and with whom does one coordinate? 
    • What kind of results does one expect to see, how are the results going to be reported and what kind of format is to be used?
    • Is the sample fit for use? (i.e. accountability, storage, seal, damage or integrity issues, lack of 702(b) portion).

    If these questions cannot be answered, seek help and discuss the issues before proceeding with the analysis. Information from the investigations branch can be obtained through  the supervisor; sample investigators may have valuable information not always present on the C/R, (e.g. photographs).     
     
    The Observant Analyst:  If the questions can be answered, then, the analyst is ready to begin the analysis, with this added note of caution. Regardless of the stated or implied objectives of the C/R or the analysis chosen, the FDA analyst is always to be alert for unexpected developments. The purpose of regulatory sample analysis is to discover evidence of a violation of one of the various laws enforced by the agency. Many routine analyses have taken a sharp change of direction due to a chance observation by an alert analyst. Each analysis represents a new and potentially provocative situation that challenges the analyst's powers of observation and scientific curiosity. An alert, inquisitive approach to sample analysis is every bit as valuable to the agency as any scientific expertise the analyst may have.

B. Scale of the Analysis

Macroscopic and microscopic procedures for characterizing defects in foods tend to supplement each other, and together provide a comprehensive evaluation of defects in the product. It is important that the analyst realize the close association of the macroscopic and microscopic methods for use as a joint approach in solving analytical problems

  1. Macroscopic Methods of Analysis

    To consumers, "macroscopic" analysis of a product refers to an evaluation of the substance through the use of their unaided senses (primarily sight, smell, or taste).  Every consumer in our society who exercises some judgment in the purchase of foods and other consumer goods, knowingly or unknowingly conducts some form of macroscopic examination to detect apparent or obvious defects. The examination may range from a cursory, perhaps unconscious visual check of the product to confirm that everything "looks right", to a much more detailed scrutiny for defects. Regulatory authorities, in fulfilling responsibilities for protecting the public health and safety, conduct systematic examinations to disclose not only apparent defects, but also hidden defects. Over the years, standardized methods of macroscopic examination have evolved for determining filth, decomposition, and foreign matter. These methods of analysis have evolved with the input of producers and consumers as well as regulatory authorities.
     
    In general, "macroscopic" or macroanalytical methods for food examinations primarily depend upon the direct sensory input of the analyst.  For example, visual examinations are typically conducted with the naked eye.  These exams are occasionally supplemented by low power magnification to confirm defects observed initially with the naked eye, or to describe the defects in greater detail. 
     
    There are several major advantages to the use of macroanalytical procedures. They are inexpensive and call for little specialized equipment. They generally permit the analysis of a large quantity of product in a relatively short period of time, thus allowing the analyst to assess the overall condition of the lot quite rapidly. The analyst can quickly identify and isolate those portions of the lot which may contain defects and thus limit the amount of material which may need a more detailed, microscopic evaluation. 
     
    Although macroscopic methods have many positive aspects, they may not be the method of choice for every analytical situation. In fact, the very features which add to their usefulness may also limit their application in some situations. Because macroscopic procedures deal with defects which are discernible to the unaided senses, they are not usable for defects hidden from the senses such as those defects too small to be visible to the eye, or those obscured through processing or other factors. In such cases, microscopic methods are essential for characterizing and evaluating the defects in the sample.
  2. Microscopic Methods of Analysis

    Microscopic methods of analysis involve the detailed examination of a very small portion of the sample; these procedures provide a different type of information than macroscopic methods. They are used to describe and quantify defects on a different scale than macroscopic methods, and to identify "hidden" defects that cannot be detected through a gross evaluation of the sample. However, microscopic methods also have limitations; they tend to be more time-consuming and more expensive, and they need more specialized equipment. Also, because they are limited to the analysis of a very small sample, the results are not always representative of the overall condition of the lot, thus representative sampling plays a more critical role in this type of analysis.

C.  Method Selection for Filth Analysis

SeeORA Lab Manual, Volume II, Section 5.4, on Test Methods and Validation.

  1. General information

    Filth methods commonly used have been published in the AOAC Official Methods of Analysis or the Macroanalytical Procedures Manual. AOAC methods employed by FDA analysts have been proven to give reliable, consistent results; these methods have been validated through collaborative study. If possible, the analyst should use official methods whenever they are found, as written and without modifications. 
    1. Association of Official Analytical Chemists (AOAC)

      Many of the analyses in the following sections will be found in the Official Methods of Analysis published by AOAC INTERNATIONAL (AOAC).  The analyst should become familiar with the editorial conventions of this text. All editorial conventions are described in the front of the manual.  Boldface reference numbers in the text of a method refer to safety precautions, apparatus, or other critical information. The entire method should be read and each reference looked up before beginning the analysis. A star after the title of a method indicates that the method will be dropped from the next edition, as "Surplus". "Surplus" methods can be used, but if one of these methods is used, the analyst should inform the AOAC Section Editor, so the surplus decision can be reconsidered. Journal references at the end of each method refer to the article that reported the results of the collaborative study for that method. These journal articles often contain useful additional information, such as problems encountered and expected recoveries. Formulae, such as (2+1), indicate volume ratios for mixing the reagents being discussed.  
       
      The AOAC's chapter, "Extraneous Materials: Isolation," initially discusses general techniques, apparatus and reagents. It also cites how to record and report results, and gives counting/identification instructions. This information is important and should be read by the trainee before the first attempt to use the manual; use the most current edition found.
    2. Macroanalytical Procedures Manual
       
      Also known as the FDA Technical Bulletin Number 5, originally published by the AOAC, this publication is now out of print, but has been placed on the FDA intranet at: http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/MacroanalyticalProceduresManualMPM/default.htm. The web version does include some minor changes (error corrections) that are not reflected in the printed versions. The web version also includes web citations to the Compliance Policy Guides for products, making it easier to find related guidance.
       
      This one volume manual compiles and organizes the standardized methods of macroscopic analysis which are useful in determining defects in various types of foods. Although in a general sense, the term "macroscopic" is not as broad as the term "macroanalytical," for the purposes of this manual, the terms are used interchangeably. 
       
      This manual compiles standardized macroanalytical procedures for identifying defects in food products. However, macroscopic procedures are frequently interrelated with and supplemented by microscopic ones, each providing the analyst with different types of information. For this reason, the Macroanalytical Procedures Manual will refer to microscopic procedures in some situations. 
       
      These microscopic procedures may be grouped into three categories:
      • Microscopic methods which have been published by the AOAC in Chapter 16 ("Extraneous Materials") of the Official Methods of Analysis. Where needed, this manual simply refers the analyst to the applicable section of the AOAC volume for the correct method. 
      • Microscopic methods which have been published in the AOAC volumes, but which are adapted by the analyst for a particular situation. In these cases, special instructions are provided in this manual so that the analyst can modify the microscopic procedure as needed. Reference is made to the correct section of the AOAC. 
      • Microscopic procedures which have been developed and are in use, but have not been subjected to collaborative study and thus are not yet published by the AOAC. These procedures are included in full in this manual so that they are not lost to the analyst. 

      Thus, when using this manual, the analyst may be instructed to combine both macroscopic and microscopic techniques. Examples of this can be seen in the method for determining decomposition in frozen strawberries, which utilizes macroscopic "pick-out" of defects (see Chapter V, Section 9.N.(4)b.) supplemented by the microscopic mold count technique (Chapter V, Section 9. N.(4)c.). Information provided by the microscopic techniques will aid the analyst in interpreting and evaluating the macroscopic findings and in determining the overall quality of the food.

D. Sample Contamination

Read and follow the local laboratory SOP's dealing with cleanliness and quality control in operation.

The filth analyst exercises constant vigilance against the inadvertent introduction of any type of outside contamination into the sample(s) under the analyst's care. This vigilance extends to those who work in the laboratory if their work can contaminate the sample.

  1. Techniques to Prevent Sample Contamination
    • Glassware and other equipment are kept scrupulously clean and stored in clean, enclosed areas when not in use. Equipment should be given a preliminary "extra" cleaning prior to analysis by rinsing with clean water or wiping with clean towels. Purity of reagents are assured by proper storage, and filtering.
    • Steps are taken to assure that the analyst's person or clothing do not contribute anything to the sample. A clean laboratory coat is essential. The analyst develops personal techniques of handling samples and equipment that avoid bodily contact with the sample or sample contact surfaces. Spoons, scoops, tongs, and rubber gloves are useful for this purpose. Hair (both human and pet) and lint are the primary contaminants to be guarded against. Precautions such as these combined with personal hygiene will preclude any possibility of analyst-related sample contamination.
    • During analysis, the sample be is protected from airborne contamination as much as possible without interfering in the analytical procedure. Containers such as trap flasks, beakers, and percolators that are to be left standing for a period of time should be covered. Petri dishes are employed in the conventional manner, with the larger diameter part used as the top or lid for storage of filth extraction papers. Bench tops and other surfaces should be cleaned to avoid dust buildup, and reagents should always be stored in capped or stoppered vessels.
    • Guard against cross-contamination between two samples or two sub-units of the same sample. The same piece of equipment is never used twice without thorough washing. Unused portions of reagent are never returned to the original container, but disposed of in a proper manner. Spatulas, tweezers, and other implements are cleaned before they are used to manipulate samples or reagents. Any reagent whose purity is in doubt is disposed. Aerators and wash bottle nozzles should never come in contact with any sample or piece of equipment. If this happens, a thorough cleaning is needed. These and other common sense rules are followed to protect the integrity of the results of analyses.

E. Use of Quantitative Transfer

The concept of quantitative transfer applies to each sample portion from the moment the sample container is opened until the final results are reported.  This demands that every vessel, implement, and operation be considered a possible cause of sample loss.

Each time an analytical portion is transferred from one vessel to another, every effort is taken to assure that no amount of material, however small, is inadvertently left behind in the old vessel. While some small loss appears inevitable in every transfer operation, the analyst can take steps to keep this loss to a minimum.

  • For methods using wet chemistry, the wash bottle is the analyst's most versatile tool for accomplishing quantitative transfer. Many methods specify wash bottle liquids for optimal recovery of filth, and care is taken to use the correct wash to avoid interference with the extraction process.
  • The analyst also avoids losing small amounts of sample through dripping, splashing, or leakage. Once a loss has occurred, there can be no replacement or compensation, thus the results of the analysis may be compromised.
  • Chipped or cracked vessels and overzealous heating (bumping) and sloppy or overpressure sieving operations should be avoided, as these have the greatest potential for sample loss. Sieves are also inspected for rips or deformities that could result in losses.

F. Apparatus Selection

For each analysis, the analyst chooses the proper apparatus. Often the analytical method will call for explicitly described apparatus. Regardless, the analyst is to be aware of a few general restrictions on apparatus.

  1. Avoid sample contact with plastic vessels and implements.  Hairs and insect fragments will stubbornly adhere by static force or surface texture to plastic surfaces resulting in reduced filth recovery.
     
  2. Do not use chipped, scarred or broken equipment. Irregular surfaces can snag material, causing problems.
     
  3. Do not use glass equipment when analyzing a product for glass contamination.
     
  4. Use plain weave (not twill weave) sieves. After each use, sieves should be backwashed and inspected for tears or deformities. Fine mesh sieves should never be touched by spoons, scrapers, etc. The most drastic cleaning allowable for fine mesh sieves is soaking in mild cleaning solutions such as bleach, pancreatin or soap. "Clogged" sieves should be discarded if they cannot be cleaned, or the fabric should be replaced.

G. Measurements

A complete description of the filth found during an analysis always includes the sizes, in metric units, of the filth elements. This important factual evidence should never be omitted.  The analyst should use the correct number of significant figures indicated by the method in the measurement of filth elements, reagent amounts, net contents, etc. This will help avoid overstatement or understatement, rounding errors, and will increase precision.

See ORA Lab Manual, Volume III, Section 7, on Statistics.

A complete, accurate report of the analyst's work (not just results) is essential. Agency and court decisions may be based, in part, on the analyst's written report of the analysis and reported results.  The results should be reproducible and the analyst's report contains an account of every operation performed and by whom, every result, and evidence of sample continuity. During the next phase of training, the trainee will be evaluated not only on performance of analytical methods, but on reporting of analyses as well. Reports should be clean, well organized, and neat.

H. Laboratory Safety

Before working in the laboratory, the trainee should have laboratory safety training.  Prior to starting an analysis, the analyst carefully considers the hazards that could be presented by the equipment and reagents in the analysis, and plan measures to avoid or minimize these hazards. The analyst should have read the MSDS sheets,  and be aware of the location  and use of the emergency showers, eyewash stations, first aid kits, safety hoods, personal protection equipment, and other safety or emergency equipment in the laboratory.  Constant caution and care will assure a safe, accident-free working environment.

During the next phases of training, the trainee should always discuss beforehand with the trainer the potential hazards involved in the proposed analysis and the precautions needed for a safe analysis.

See ORA Lab Manual, Volume III, Section 2, on Environmental Health and Safety.

I. Assignments

Read the following:

  1. Chapter 16, Section 16.1.1 and 16.1.2 of the current edition of the AOAC Official Methods of Analysis and look at the general organization of the methods.
  2. Preface and Introduction in the MPM and look at the general organization of the manual.
  3. The lab's Standard Operating Procedures that relate to the filth lab.

 4.4.2 Visual and Macroscopic Methods

A. Objective

The trainee learns and performs various common food analyses for gross contamination, and reports analytical results on the official forms, especially the analytical worksheet.

B. Discussion

Visual and Macroscopic methods examine relatively large amounts of product for contaminants easily detected with the unaided eye. A portable magnifying lens or magnifying lamp may be used, but the magnification range should not exceed 5X for general purposes. Lenses of higher power have restrictive fields of view and impractical, short focal working distances. If the analyst suspects the contaminants are likely to be much smaller than a poppyseed, or otherwise difficult to detect, then reliance should not be placed solely in a visual examination. A stereomicroscopic method should also be used to evaluate the contamination.

  1. Sample Preparation
    1. Sieves

      Dry sifting is a common way of separating macroscopic contaminants from a large bulk of product. The material retained on a particular sieve is referred to as "overs" and the material passing through the sieve as "throughs." One versatile feature of analytical sieves is that they can be nested in order of increasing fineness so various sizes of particles can be separated out in one operation. Sieve mesh sizes are indicated by a standardized number scale, with the larger numbers denoting successively finer meshes. In general, standard Number 8 mesh sieves are used to retain larger rodent fecal pellets, gross contaminants such as sweepings, or product units the size of a small pea or larger. Standard Number 20 mesh sieves will retain most adult insects, many larvae, and smaller rodent pellets. Sieves that are finer than Number 40 mesh are usually too fine for macroscopic applications. Between operations, these sieves can be cleaned using an air current and a dry, clean towel, or they can be washed. Also note that for some products, like peppercorns, special sieves have been designed to sift and grade the product and to determine the "fines" in the finished product.
    2. Jones or Riffle Divider
       
      This device can be used to thoroughly mix products such as wheat kernels or coffee beans. Passing the sample six times through a Jones divider produces a totally random distribution of product units. Similarly, it can be used as a sample-halving device; the device halves a sample on each pass through the divider. A portion of product can be isolated from the main bulk by passing through the Jones divider and discarding successive halves until the desired portion size is approximated.
    3. Seedburo Grain Inspection (Picking) Tray and Cover

      Commonly used in USDA Grain Inspection Services, the lower unit consists of a metal tray with linear groves in which the product rests. The top is also a tray, but with a compressible foam pad. Spread and examine the product on the bottom tray, then when finished, add the top tray, compress, then invert the two trays together, and remove the (now, top) grooved tray. This allows one to see the opposite side of the materials. It helps insure that all sides of the individual grains are examined without having to turn each piece individually.
    4. 20" Wide Roll of White Butcher Paper
       
      Paper on one side, plastic coated on the other—this inexpensive paper is both strong and large enough to examine almost any sample. Used plastic side down, it can be cut to whatever length needed, and when replaced, (i.e. between each subsample analysis), it insures subsample integrity and a new clean working surface. Samples can be shaken into a singular flat layer, and then the items can be manipulated as individuals, or grouped together. The paper can be folded, or closed, to accommodate easy transfers back to the original containers. Wet samples can also be examined by using the plastic coated side.
       
    5. Pharmacist's Tablet or Counting Tray
       
      This small 6" X 8" tray has a flat surface with a round grove on one side of the tray. The grooved side has a matching cover that flips into place to cover the grove. Tablets (or in our case, beans, peas, nuts) can be counted (in groups of five or ten) and slid into the groove until the desired number is reached, then the lid closed, extra product removed, and the counted units poured out for closer examination or weighing. It can be used for estimating product weight versus count, and for sequential sampling purposes.
    6. Examination Area
       
      It is recommended that a 2' deep by 4' wide area be set aside that is clear of obstructions and superfluous equipment. It should be easy to clean, preferably with a white or wheat colored matte surface. The area is to be well lit, at 150-200 foot candles, preferably with natural colored, shadow and glare free adjustable lamps. Ideally, the tabletop height can be adjusted to the analyst's need and will be open underneath for knees, stool or chair work.
    7. Interpretive Line System
       
      This is a series of photographic slides or photographs originally developed by USDA for their GIPSA grain inspection service. These materials are purchasable from Seedburo, and provide exemplars of the types of damage that may be seen in various products including barley, beans, corn, lentils, peas, rye, safflower, sorghum, soybeans, sunflower, wheat and other products. It should be used in combination with FDA authentics, toxic and weed seed identifiers, and the FDA slide series.
    8. Other Tools
       
      Various spoons, spatulas, knives, flour slick, small brushes, pans, trays, fine and course forceps, needles, and various sized petri dishes. 

C. Assignment

  1. Perform the indicated visual and macroscopic analyses under the direction of the trainer. (Substitutions may be made based on product availability, preserving the type of analyses presented)
  2. Report the results of each analysis using the correct forms and formats.
  3. For each analysis, discuss with the trainer:
    • the method used and difficulties encountered
    • results and their significance 
    • the quality of reporting

D. Analyses

  1. Whole cereal grains (Macroanalytical Procedures Manual (MPM), Chapter V.3.A.). The analyst is to pick out whole insects, webbing, fecal pellets, extraneous material, and kernels damaged by heat, mold, and/or pests. Good lighting and a light-colored, non-reflective background are needed. Insects cause damage to kernels either by surface feeding or by tunneling. Rodent-gnawed kernels can be recognized by the scalloped edges, a result of the rodents' paired gnawing teeth. Use of a Jones divider may be needed.
  2. Green coffee beans, dried beans, or lentils (MPM, Chapter V.1.A, V.11.G.). For bulk sampled product, a Jones divider is useful for mixing or compositing as well as for obtaining the analytical portion. The trainee looks for insects, insect damage, and mold. Mold is confirmed microscopically, by observing hyphae and/or fruiting bodies. If possible, the grading of coffee can be demonstrated by a trainer. (FDA By-lines 7:285-91, May, 1977.) Lygus bug damage to beans should be demonstrated.
  3. Flour (MPM, Chapter V.2.B). The flour is sifted portion wise through a standard # 20 mesh sieve. Examine sieve "overs" for filth. The portion size is generally the entire contents of a consumer package. Sieve "throughs" may be saved for additional analysis in the next section.
  4. Whole or crude spices (MPM, Chapter V.8.). Analysis of spices often entails breaking or cracking open a large amount of product in search of insects or signs of insect activity. Rodent defilement may also be encountered. A special sieve is used for peppercorns, over which the product is passed a specified number of times. The analyst should check beforehand whether the product, especially peppercorns, has been fumigated for bacterial contamination. Cinnamon or Cassia sticks are cracked for internal mold.

    Safety Note:  Precautions and clean-up for Salmonella-type organisms may be indicated if there was no fumigation. Mite infestations appear as "moving" dust or surface feeding on inner or protected surfaces. Feeding surfaces are sometimes discolored, with adhering, fluffy, granular material (cast skins) often encountered in surprisingly large quantities.
  5. Whole figs or dates (MPM, Chapter V.9.F). This type of analysis may call for a statistically-derived sequential examination plan that is faithfully executed for valid results. A sharp knife is needed to cut the fruit since the insect contamination usually occurs near the center or pit. An occasional small wasp may be found in some types of figs. These insects are tolerated since they are essential to develop and pollinate the fruit and they enter into the fig to complete their life cycle.
  6. Shell nuts (MPM, Chapter V.10.A). For this analysis, a sturdy hammer and pounding block are helpful. The various types of reject nuts are described in the method. Mites can sometimes enter pecans or walnuts through breaches between shell halves and can build up impressive populations inside the nut, causing considerable damage.
  7. Cocoa beans (MPM, Chapter V.4.A.).  Mold should be confirmed microscopically.  The trainee should discuss with the trainer the best technique for mixing the sample, considering all factors including size of sample, size and number of subsamples, administrative guidance, etc.
  8. Blueberries or cherries for maggots (AOAC Official Methods of Analysis, current edition, Chapter 16 on "Extraneous Materials: Isolation;" or MPM, Chapter V.9.C or D). Preliminary manipulation of the product is needed in order to free the maggots from the fruit tissue. Other insects may be encountered, as well as decomposed berries. If substantial decomposition is suspected, additional analysis may be indicated. Maggots are preserved in dilute (60-75%) ethanol. 

 4.4.3 Microscopic Methods

A. Objective

The trainee will learn how to perform the most common types of analyses for "light filth" in foods using various flotation techniques, examine the recovered material for "microscopic filth," and report the analytical results on the worksheet.

 4.4.3.1 Flotation Techniques

A. Flotation Techniques

Flotation methods are designed to isolate microscopic filth by floating the filth upwards, typically in an oil/water-phased system. Insect fragments, mites, and hairs are lipophilic and like to be in the oil phase, thus they float to the surface with the oils, (hence the term "light filth").  Plant tissues and most related tissues are hydrophilic, and they prefer to stay in the water phase. Common gravity further helps this process, and larger particles sink. To accomplish the separation of filth from food, use a number of solution systems to insure that the majority of the product sinks, while the oils with trapped filth, floats. Often, the analytical portion undergoes a pretreatment, to enhance this effect. The analyte (filth) portion is usually very small, both in a weight to weight relationship to the food (parts per million) and in size or scale. Typically, the recovered filth contaminants are examined under a widefield stereomicroscope. Once found, the filth items may have to be mounted for microscopic identification, thus the term "Microscopic examination".

Most microscopic methods are found in Chapter 16 of the AOAC "Official Methods of Analysis," consisting of a compilation of validated methods. The trainee should be familiar with the initial information concerning reagents, apparatus, and techniques, and the safety precautions referenced parenthetically in the text of each method.

All microscopic methods begin with the analyst weighing out a prescribed amount of material to be analyzed. This step is critical because the guidelines for how much filth can be in a violative sample, is based on a set amount of product being analyzed.

From this point on, the exact steps followed may vary significantly from product to product, and are prescribed in the method; the method is to be followed precisely.

Variations in methodology based on product

  • There may or may not be a pretreatment step to remove excess fats and oils found in some foods (e.g. spicy sauces). 
  • A digestion step is often used to hydrolyze or digest the product into small particles (e.g. - bread). Some foods, once digested, are wet sieved, that is washed with very hot water over a fine sieve, to flush away excess product and oil.
  • Some products may need a hydration, to swell, add water, or saturate the product (e.g. raisins)
  • Alcohol may be added to the water, to help saturate the product, penetrate the skins, and dissolve or solubilize excess fats or oils.
  • Detergents may be added to loosen the filth, to saturate the food, to emulsify or trap the oils.
  • Compounds such as salt or EDTA may be added to help products sink or make them heavier, or to make the water phase even heavier.
  • Light hydrocarbon products may be added or the choice of flotation oil changed to help capture the lipophilic filth.

There are basically two physical systems to use in extracting light filth from foods, the Wildman trap flask and the corning percolator. The Wildman trap flask is a closed system, that is, the volume and composition of the aqueous phase remains constant. The filth laden oil interface layers are captured in the neck of the flask. Utilizing a rubber disk on a rod, the oil and interface layers are cleanly separated from the aqueous phase and removed (poured off) from the trapping system. The corning percolator is an open system which allows for repeated drain and refill cycles further isolating the oil filth interface layer. The final drain of the percolator results in a totally isolated oil phase with just a very small amount of the aqueous layer left.

The final step of most flotation methods is to transfer the oil and extracted filth to a ruled fast draining filter paper for microscopic examination. For this we use a Buchner funnel- fitted into a large side arm flask attached to a vacuum pump. A Buchner funnel is an open top funnel, with a porcelain screen about 1 cm below the opening. A 60 mesh brass or stainless steel fine screen is cut to fit over the porcelain openings. This screen serves to evenly distribute the vacuum under the filter paper and to help distribute the product over the filter paper. The filter paper is wetted, and then centered over the opening, and the vacuum draws the paper down into the funnel forming a small cup-shaped filter. The trapped filth and oil is poured into the filter paper cup and the recovered tissues and filth are evenly distributed over the filter paper. Each extraction paper is then placed in a petri dish bottom plate, and glycerin alcohol, a (1 + 1) mixture of glycerin and 95% ethanol, is added to partially wet the material on the paper, but not to the point debris can float freely over the paper. The edges of the filter paper cup are laid down, and the lid is placed on the dish to keep out dust and minimize evaporation until the extraction paper can be examined. Each petri dish is immediately labeled with sample, subnumber, date, and the analyst's initials. Extraction papers become part of the sample reserve and should be carefully preserved. Refrigeration or the addition of a few drops of formaldehyde are effective preservatives after the papers have been examined.

Note: The petri dish should not be inverted, with the filter paper in the larger diameter top, covered by the small diameter piece. This allows airborne contaminants to contact the filter paper and causes rapid evaporation of the glycerin alcohol. It may also cause loss of some filth by adherence to the parts of the dish that contact the filter paper.

B. Assignment

  1. Read  pp. 173-180 in: Gorham, J. R. (Ed.) (1981). Principles of food analysis for filth, decomposition and foreign matter (FDA Technical Bulletin No. 1, 2nd ed.).  Gaithersburg, MD: AOAC International.
  2. Perform the indicated analyses under the direction of the trainer.
  3. Report the results of each analysis using the forms and formats.
  4. For each analysis, discuss with the trainer:
    • the method used and difficulties encountered 
    • results and their significance
    • the quality of reporting

C. Analyses

  1. Flour (AOAC Official Methods of Analysis (OMA), Method 972.32 "Light Filth (Pre and Post Milling) in Flour (White)").  Acid hydrolysis is employed to digest the flour, and the filth is isolated by flotation in a percolator. Due to the uniformity of flour milling processes, pre-milling and post-milling contaminants can be roughly recognized by size. It is, therefore, important to record the size ranges of contaminants so that compliance personnel can accurately interpret the analytical results.
  2. Fig or fruit paste (AOAC OMA, Method 964.23 for "Filth in Fig and Fruit Paste").  Filth flotation is accomplished using a Wildman trap flask. The trainee practices quantitative transfer from the flask to a beaker prior to attempting the analysis. A smooth motion is needed to pour the trapped material into the beaker while holding the trap flask rod out to exclude the aqueous phase of the liquid system. About 1/4"-1/2" of aqueous phase is trapped off along with the oil phase to ensure that no filth is left below the stopper. While holding the stopper in the "up" position, the flask neck is rinsed with the same aqueous solution and the rinses are poured into the beaker.
  3. Chocolate (AOAC OMA, Method  965. 38 B (b) "Filth in chocolate").  This method uses a detergent to defat the product prior to the flotation.  The extraction papers (filter papers containing the extracted filth) will give the trainee a challenging exercise in distinguishing between very similar-appearing insect and plant fragments, and introduce bleaching techniques that may prove useful in other situations.
  4. High bran content bakery goods (AOAC OMA, Method 972.36 for "Light Filth in High Bran Content Breads").  This method employs a defatting step to enhance the separation of filth from product. The defatting should be performed in a hood to avoid fumes. 

 4.4.3.2 Sedimentation and other Specialized Techniques

A. Objective

The trainee will learn how to perform some common types of food analyses for various types of filth not easily recovered by flotation, and how to record the results.

B. Discussion

Three very different problems that cannot be resolved by using any of the techniques described thus far will be demonstrated in this section:

  • The first problem deals with "heavy" filth. As the name implies, these methods rely on a combination of gravity and density in a liquid system that allows the material to sink to the bottom of the container, while the lighter generally organic materials are floated off. 
  • The second problem deals with thin-skinned insects, like maggots, insect eggs, and mites. Because of their thin exoskeletons or shells, they are less oloephilic than the more mature stages of the animal, and they too prefer to sink- even in oil/water phased systems.
  • The third problem is how to count/detect maggots or mites if they are buried deep inside the plant tissues, and they are attached to tissue material?
  1. Heavy Filth

    In heavy filth methods, we are typically looking for heavy material contaminants such as sand, glass, metal fragments, and even feces. Basically, the analyst is going to make a liquid slurry of solid materials using different solutions, that, with stirring, act to both solubilize the material to loosen up the product and free the heavy materials, thus allowing the heavy materials to sink. In the past, carbon tetrachloride and chloroform were used, now solutions like chloroform alone or salts are used, which when added to water, solubilize and make the solutions much denser than water alone.  The higher the density of the solution, the greater the chance that the light weight organic material will float and the heavy materials will sink. 
     
    Keep in mind, that usually hairs and insect fragments are not recovered by these methods. The analyst often will use the heavy filth method as a prelude or preliminary step to a light filth extraction. These methods are a very good way to defat or remove excess oils from the food product. The analyst usually retains the poured off organic materials for his next analysis, which is typically light filth. Hairs and insect fragments come off in the organic material, and that material should be examined for these and other types of filth (especially fecal pellet fragments). 
     
    Heavy sediments such as glass and metal shards, sand, minerals, bone and other materials may adulterate some products. The analysis should be tailored to the particular kind of analytes suspected in the product. For example, if glass contamination is suspected, the analyst avoids using all glass apparatus such as glass beakers and stirring rods. The product's original container is always examined to determine if it could have contributed to the contamination.
     
    Once these materials are recovered, they are described.  Pictures help, but a physical description of the recovered material is annotated on the worksheet. The descriptions should be precise, accurate and brief. Although it is not possible to cover all of the characteristics or terms used in material identification, for training purposes, one or more of the following characteristics may help describe these materials (this is not an all inclusive list;  use a thesaurus if needed, and modifiers when needed).
     
    Material Descriptions: Size, shape, color, finish (matte/gloss), variegation, surface texture, surface coating, lamination, presence of a parental (original) face or markings, scoring, density, fracture, fraying, melting point, density, inclusion, assemblage, distance, interval, parallelism, obliqueness, angularity, position, curvature, softness, elasticity, voids, optical character, etc.
     
    For example, glass has many characteristics, but it can be differentiated from sand grains or other crystalline look-alikes by two principle characteristics. Broken glass exhibits acute angular, conchoidal fractures (resembling the markings of a clamshell) and it shows no color (isotropism- no optical activity) when viewed between crossed Nicols on a polarizing microscope. When combined with size, shape, hardness, density, solubility, parental face characteristics (if present), and refractive index, there is little doubt left towards the identity of the material. The analyst has to list the pertinent facts in the report and draw a conclusion based on those facts.  Keep the report brief and to the point.
  2. Maggots and Insect Eggs

    Another problem area deals with the isolation of thin-skinned insects, like maggots, insect eggs, and mites. As stated earlier, because of their thin exoskeletons or shells, they are less oloephilic than more mature stages of the animal, and they prefer to sink even in oil/water phase systems. In the olden days, people did not want to go anywhere near tomato canneries because the canneries would dump the waste tomato skins in huge piles outside the plant. This created ideal breeding grounds for flies and maggots. Some of these flies ended up inside the plant and the eggs and newly hatched maggots would be in the product. One of the first things filth analysts learn is if a mixture of oil and tomato tissue floats, then the eggs and the larva sink to the bottom of the flask. In the method below, Analyses Section D.2., the maggots and eggs are drained off, the oil and tomato tissue remain in the separatory funnel.  This method is exactly opposite of the extraction procedure in "light filth" methods.
  3. Maggots and Mites in Mushrooms

    Maggots and mites present the filth analyst with a real challenge, especially when embedded deep in the product's tissues. To extract them, the maggots and mites are mechanically freed; this is done in a blender. A challenge still exists when they are free; the maggots can not float off, nor can they sink because of the extra tissues they are mixed with. This method demonstrates staining the maggots and mites so they are much easier to see. It is a unique method that works very well for all mushroom products, and incorporates a bleaching technique that illustrates how easy it is to clear tissues, yet not affect the filth one is looking for. It also teaches the analyst to be careful in how long to blend the products and how many subs can be done in a timely manner.  Analysts should practice with training samples before receiving samples for regulatory analyses.

C. Assignment

  1. Perform the indicated analyses under the direction of the trainer.
  2. Report the results of each analysis using the correct formats.
  3. For each analysis, discuss with  the trainer: 
    • the method used and difficulties encountered
    • results and their significance
    • the quality of reporting

D. Analyses

  1. Ground spices for heavy filth (AOAC Official Methods of Analysis (OMA), Method  978.21 for "Light Filth in Capsicums (Ground), sedimentation"). The liquids selected for these methods are dense enough to float the spice material but not heavy filth such as feces, rocks, sand, and dirt. Decanting the spice material leaves the heavy filth behind to be examined microscopically before the weight is determined. The cautions indicated in the text are to be observed. Substitute Chloroform for all Carbon tetrachloride citations. 
  2. Tomato products for fly eggs and maggots (AOAC OMA, Method 955.46 "Filth in Tomato Products"). A large separatory funnel is used to separate product from maggots and fly eggs. The latter settle to the bottom and are drawn off through the stopcock. The analyst should become familiar with the appearance of fly eggs and maggots prior to attempting the analysis.
  3. Mushrooms for maggots and mites (AOAC OMA, Method 967.24 "Filth in Mushrooms").  A dye is employed to make the filth highly visible (purple) against the background of bleached (white) product. The product is blended in order to free maggots and mites that are embedded in the mushroom tissue or wedged tightly in the gills. The crystal violet dye is soluble in ethanol for clean-up purposes, but try not to get it on the hands.

 4.4.4 Mold Detection

 4.4.4.1 Gross Mold Contamination

A. Objective

The trainee will learn the general diagnostic characteristics of mold.

B. Discussion 

Most people can recognize visible mold growth by its characteristic growth habit, colors, and musty odor, without the aid of magnification. Under a low-power hand lens, however, typical mold consists of a mass of thread-like, branched filaments. The mass is called a mycelium and the individual filaments are called hyphae. Spore-bearing fruiting bodies, whose shape varies between species of mold, may also be found.
 
In most cases, however, macroscopic observations are insufficient for FDA purposes. Some non-mold plant diseases or other conditions may superficially resemble mold. Also, decomposition can sometimes mask the presence of mold. For these reasons, the analyst confirms the presence of mold microscopically. This can be done by preparing an aqueous slide mount of a small portion of the suspected mold, examining it for the presence of hyphae. Microscopically, mold hyphae can be distinguished by one or more of the following characteristics:

  1. Parallel walls.  Although individual hyphae may vary in size, the diameter of any single hypha is constant. Mold hyphae are basically tubular, with parallel walls.
  2. Septation. The presence of parallel cross walls (septa) in hyphae is a characteristic of many molds. They give the hyphae a segmented appearance. Some plant hairs may have cross walls but they are usually not parallel to each other.
  3. Granulation. The protoplasm of mold may exhibit a distinct granular appearance. This is an especially useful characteristic among larger species that may not exhibit septation.
  4. Branching habit. When present, this is one of the most reliable characteristics. Mold hyphae typically have branches that are the same diameter as the main trunk. Typical mold branches extend out at right angles to the main trunk. 
  5. Rounded ends. The natural tip of a hypha is normally rounded like a test tube bottom. Hyphae that are broken, however, typically break off squarely. 

C. Assignment

  1. Using moldy fruits or other foods, practice preparing aqueous slide mounts and distinguishing, under a compound microscope, the mold hyphae.
  2. Examine at least two samples of different whole fruits, vegetables, or spices for macroscopic mold contamination, confirming the mold microscopically. Methods for some of these products can be found in the "Macroanalytical Procedures Manual".
  3. Report the results of each analysis on the correct forms, discussing each report with the trainer.

 4.4.4.2 Howard Mold Count

A. Objective

This section describes the Howard Mold Count technique.

B. Discussion

Howard Mold Count procedures are empirical methods that are to be precisely followed in every detail for each type of product in order to obtain satisfactory results. Experience has shown that mold counting cannot be reliably learned without the help of an experienced instructor who can give the trainee personalized instruction.

  1. Microscope 
     
    A Howard Mold Count is performed on a compound microscope with certain features. The first requirement is a lens system that has a standard microscope field diameter of 1.382 mm. The eyepiece has a micrometer disk ruled in squares, each side of which is equal to one-sixth the diameter of the ocular lens opening. A Howard Mold Count cannot be performed using a microscope that does not meet these requirements.
  2. Howard Mold Count Chamber
     
    This is a specially constructed slide and cover glass unit that is used. It is designed to contain 0.03 cc of material on a central platform with the cover glass in place. The platform is surrounded by a moat and flanked left and right, beyond the moat, by shoulders whose height is 0.1 mm taller than the platform. The combined exacting requirements of the microscope and Howard Mold Count chamber ensure that the analyst, at all times, views a precisely known amount of product. Each Howard Mold Count chamber has a scored calibration circle of 1.382 mm diameter, or has scored parallel calibration lines, 1.382 mm apart, to use to check the standard microscope field diameter.
  3. Sample Preparation 
     
    The product is prepared exactly as stated in the method for that product. The sample is thoroughly mixed both before and after dilution. Immediately before each slide is prepared, the sample again is thoroughly mixed. This is important to assure uniform suspension of mold filaments.
  4. Slide Preparation  
     
    For each preparation, the Howard Mold Count chamber is completely clean and dry. A clean scalpel is dipped into the well-mixed sample and then touched against the platform of the Howard Mold Count chamber so that just enough sample is transferred to fill the platform when the cover glass is in place. The drop of sample is evenly spread using the scalpel, and the special cover glass is lowered over the platform until it almost touches the product with the cover glass sides aligned with the shoulders. The cover glass is quickly pressed down, spreading the sample evenly over the platform and avoiding the trapping of air bubbles under the glass. A proper preparation has the platform entirely filled with the product, no air bubbles, and no spillage into the moat. If a proper preparation is not obtained, the entire chamber should be cleaned, dried, and another attempt made.
  5. Newton's Rings 
     
    Newton's rings are a rainbow-type optical phenomenon produced between each shoulder and the cover glass when they are in contact without applied pressure. The rings are observed by holding the completed preparation at a slant so that light is reflected off the cover glass. The presence of Newton's rings assures that the depth of product on the platform is 0.1 mm. Their absence indicates that either the slide was not thoroughly cleaned and dried or that product solids thicker than 9.1 mm are holding the cover glass above the designated height. In either case, a new attempt is to be made with a clean, dry slide and cover glass.
  6. Examination of the Slide
     
    After the slide is placed under the microscope, it is brought into focus and the field examined. The fine adjustment is used to bring into view mold filaments that may be at different depths of the field. The method calls for a field be counted as positive when the aggregate lengths of not more than three filaments of mold exceed one-sixth the diameter of the field. One-sixth the diameter of the field is not enough to be counted as positive: the aggregate length exceed one-sixth the diameter of the field. The drop-in eyepiece micrometer disk, divided by etched lines so that 36 equal-sized squares are formed and any side of which measures one-sixth of the field of view, should be standard equipment for mold counting.
     
    In the examination of a slide for mold, the field should be selected in a consistent manner. One method is to begin at what appears, through the microscope, to be the upper left portion of the counting area and go straight across, skipping every other field, then drop down approximately two fields and, reversing the direction, again cross the counting area, continuing this back and forth until 25 fields are examined. Fields to be counted should be selected at random. Under no circumstances should fields be selected for counting because they do or do not contain mold.
     
    If it is readily observed that the field is positive, it should be so recorded and study of the next field begun. If not enough mold is observed at first glance, the field should be carefully examined and the fine focusing adjustment used to bring different depths of the field into focus. In some instances, the mold can be seen better by changing the light intensity. A systematic search of every part of the field is needed before it can be concluded that a field is negative. When branched filaments or clumps of mold are found, the length of one filament is considered as the sum of all the branches or the sum of all the filaments in the clump. The fruiting heads, such as those of Alternaria, with any attached mycelia are counted as mold filaments. If the examination of a field reveals a piece of suspect material extending into the field from the edge, the material should be traced back so that its true identity can be established.
     
    However, only mold found within the field should be considered in determining whether the field is positive or negative. Should the identity of any filament be in doubt, it may be studied at a magnification of approximately 200X, although the length is determined at 100X. Unless the suspected filament is unquestionably mold, the field should be counted as negative. Small air bubbles, which in aggregate do not exceed one-sixth the diameter of the field, may be disregarded. Occasionally, a field is largely obscured by a mass of opaque material or air bubbles. In this case, if a count cannot be determined, the analyst should move on to the next field.
  7. Calculation of Results
     
    The results are calculated from the findings on examination of 25 fields from each of two or more slide preparations. Because comminuted fruit and vegetable products are mixtures rather than solutions, mold filaments are not always uniformly distributed among the plant fibers and tissues in the individual droplets used for slide counts. Therefore, the count of several slides of the same sample may vary, even though the slides are prepared and examined with the greatest care. Studies of deviation in mold counts indicate that the results are grouped about the average in the same way that other mixtures follow the rules of random distribution. This may account for the occasional wide variations of results. 
     
    A general rule is that two counts from the same sample should check within three positive fields; otherwise, two or more additional slides should be examined. For greater accuracy, more fields may be counted.  

C. Assignment

  1. Read  pp. 191-200 of: Gorham, J. R. (Ed.) (1981). Principles of food analysis for filth, decomposition and foreign matter (FDA Technical Bulletin No. 1, 2nd ed.). Gaithersburg, MD: AOAC International.
  2. Perform a Howard Mold Count on at least two types of tomato products and one type of berry product under the guidance of an experienced trainer. Use the correct AOAC method for each product.

D. Evaluation

With a sample provided by the trainer, perform a Howard Mold Count. The trainer performs an independent analysis to compare with the trainee's results for the evaluation of performance. The use of a compound microscope with a dual-viewing body is recommended so that the trainer and the trainee can view the sample at the same time.

 4.4.5 Analysis of Factory Filth Samples and Filth Analytical Worksheets

A. Objective

This section describes the complete analysis of a factory filth sample.

B. Discussion

New Hire Analysts should have already reviewed the ORA U site on the preparation of Analysts Worksheets and should have discussed this with their trainers. Filth worksheets follow the same format and rules as all other worksheets follow, differing only in the final content and formatting. To some extent, they are easier to complete, yet more complicated in content. This section provides guidance and practice in describing what initially appears to be very complicated samples, but in reality are relatively simple exhibits that need to be broken down into component parts. Also, submit photographs to help document findings and help others visualize in their minds what is being described.

For most exhibits, a generic method statement, similar to the following, covers all the analytical methods:

  • Methods:  Visual, Macro and Microscopic Examination, with pick out, for Gross Filth. For the Confirmation of Rodent and Bird Adulteration, See FDA Bylines #3, Nov. 1970, pp. 153-164, with method up-dates in the AOAC 17th edition, Chapter 16, with Supplements. See also FDA Technical Bulletin #1 Chapters 9 and 13 and FDA Technical Bulletin #5 Macroanalytical Procedure Manual Chapter V Parts 2B and 3A(4)(B) and Chapter VII Part 4. See also Zimmerman, M.L. and S.L. Friedman, 2000, "Identification of Rodent Filth Exhibits", Journal of Food Science, Vol. 65 (8): 1391-1394, and "Insect Penetration through Packaging Material" (AOAC 16th 16.15.05 973.63). Exhibits placed in labeled petri dishes for examination. Insects examined as above, using visual and macroscopic exam. Where applicable, see Results below for method citations as they are used in the analysis.

This is followed with the Results headers shown below, with the Sub description being a direct quote from the C/R continuation sheet followed by the analytical recovery:

  • Results-
    Sub #                      Sub Description and Filth Recovered
    -----------------------------------------------------------
                                    From C/R- "…."
                                    Sub consists of …and analysis confirms….

For each subsample, the analyst identifies the subsample and the analysis called for (rodent, insect, or other filth, some or all of the above.). This is done by visual or stereoscopic examination and confirmation of the subsample's description versus the investigator's collection report. Begin by physically segregating the filth into general categories, being sure to capture loose elements first. The type(s) of analysis to be performed will be dependent on this information; do not limit the analyses to those requested on the C/R.  Often, additional items of "filth" are found; include these items in the description.  For example, the investigator has identified rodent adulteration, but the investigator may not have seen or identified the mites or webbing from moth larvae also present. Also, keep in mind the differences between 402(a)(3) and (a)(4) evidence, and where needed highlighting (a)(3) evidence, as it could be a separate charge within the sample.

Consideration should be given to the preparation of the exhibits for use in the courtroom.  These exhibits may be displayed or passed around; prepare these exhibits for optimum viewing, yet present no hazard to those handling the exhibits.

Safety Caution:  Generally these are grossly contaminated samples and the analyst should use caution and protect themselves in preparing the exhibits. Personal Protective Equipment, ventilation and preparation sites should be located and used to prevent aerosol release of potential harmful agents or fumigants.

Whole insects

In addition to identifying the insects, the analyst should record the following: 

  • Counts or approximations- Count an area and estimate the number with multiplication factor - do not use "too numerous to count" (TNTC) 
  • Record if the insects were dead or alive.
  • Record stages of growth present.
  • Record evidence of fumigation or preservation by the inspector (This should be included on the C/R, but mistakes happen and they should be noted.)
  • Pliability, moistness, presence or absence of body fluids if it adds information regarding the relative age of the contamination.
  • Associated material (adulterated product, fecula or pellets, cast skins, etc.)
  • Size ranges.

Pellets and other excrement

  • Origin or source are identified, i.e. size and range, shape, presence of mucous coating and/or hairs, odors (weak, strong, fecal or urine-like), constituent make up. If applicable, confirm using chemical confirmatory tests. Size ranges are recorded. 
  • Age is difficult to determine but can be estimated, note if pliable, moist, insect damaged, bleached or discolored, or if brittleness is present.

Urine stains and bag cuttings
 

  • For all layers, mark and identify the interior and exterior surfaces. Note approximate size, shape, layers and construction, e.g., "4 layer kraft paper bag cutting with inside 3rd layer plastic bag liner".
  • Look for loosely adhering filth (hairs, mites, insects, etc) stereoscopically.  Pick them off, prepare and identify. Note their presence. 
  • Look for visible stains. Note size, shape, characteristics. 
  • Switch from visible light to Long Wave Ultraviolet black light, and in pencil, accurately outline the fluorescing stains. Note if the stains penetrate and how far. Note size, shape, characteristics.
  • Product beneath stains may also be contaminated, as evidenced by fluorescence, caking or lumping, or adherence to the packaging.
  • Finally, select the most characteristic stains and perform the chemical tests needed to confirm the presence of mammalian urine and its source, human, mouse, or other mammal.

Gnawing

In addition to the gnawed hole itself, adhering hairs, pellets, or urine stains may be found at or near the gnawed site. Note and confirm these as above. Gnawed packaging or product should be examined macroscopically and characterized as rodent or insect gnawing. Rodent gnawing has a typical serrated appearance (i.e. paired crescent-shaped cuts, with double incisor tooth marks). Insect gnawing, exhibiting liner striations, may also exhibit webbing, pellets, cast skins, setae, etc.

  • Record minimum-maximum diameter of each hole or gnawed area and location
  • Record direction of penetration of gnawed packaging (terracing) AOAC 17th ed. 973.63.
  • Record whether or not gnawing penetrated packaging completely
  • Record adhering product

Dead Animals

Occasionally dead rodents or related materials may be collected as exhibits. As with insects, these items need to be identified following traditional mammalian taxonomy procedures. They may also yield additional forensic information such as the presence of parasites (CDC washing and combing procedures,) and decay, desiccation, or putrefaction stages can be estimated. The references cited below Section D can help in this work.

Floor sweepings, trash collections

These exhibits may contain all of the above, and more. The easiest procedure is to take a picture, then segregate out the important or significant items that may not be evident in the picture. A simple inventory of these items is typically sufficient, and unimportant items can be lumped together as "waste paper, product, and/or debris"

Product and packaging blanks

Product and packaging blanks (uncontaminated "control" portions) should have been included in the factory filth sample. These should be analyzed in the same manner as contaminated material. If no blanks are included in the sample, the analyst derives them from the uncontaminated portions of a subsample or by securing other credible blank materials.

702(b) portions

These should be set aside and not analyzed; the reserve samples are required by law. When returning the sample to the sample custodian, consider creating or segregating the 702(b) portion. It should be in a clearly identified sealed package and the contents fully described on the C/R. This will save a considerable amount of time should the firm request the 702(b) portion.

Reporting the Reserve sample

The reserve sample is described completely on the analyst worksheet. Quotations of all identifying labels prepared by the analyst are included. The analyst preserves as much of the sample as possible in its original condition.

Fumigation or preservation applied by the analyst is noted. Finally, the seal applied by the analyst is quoted and the disposition of the sealed sample is stated. Normal operating procedure is to return the sample to the sample custodian and the date need not be stated, however special storage instructions should be pointed out on the worksheet and made clear to the sample custodian.

See ORA Lab Manual, Volume II, Section 3, Chapter 5.8, on Handling of Samples.

C. Assignment

  1. Read Phillip DeCamps November 1970 article "A guide for the examination of rodent filth exhibits and related samples." FDA By-lines No. 3, pp.153-164. 
  2. Read Zimmerman, M.L. and Friedman, S. L. (2000). "Identification of rodent filth exhibits." Journal of Food Science, Vol. 65(8): 1391-1394.
  3. Under the guidance of an experienced analyst, analyze a factory filth sample. (Note:  If the analyst has not received the training for chemical confirmation of rodent adulterants; the trainer will have to provide this training as needed.) 

D. References and Additional Method Citations

Filth Exhibits: Urine

  • LWUV light, Urine Stains on Food & Containers (AOAC Official Methods of Analysis (OMA), current ed., Method 945.88).
  • Urease Test for Urea (AOAC OMA, current ed., Method 942.24).
  • Xanthydrol test for Urea (AOAC OMA, current ed., Method 959.14).
  • (AOAC OMA, current ed., Method 959.14, Xanthydrol test for Urea, modified to include 4 µg + urea response requirement from J. AOAC Intl. 81(6): 1155-1161).
  • Magnesium Uranyl Acetate Test for Urea (AOAC OMA, 15th ed. (now surplused), Method 963.28).
  • Urease Bromothymol Blue Agar Test for Urea (AOAC OMA, current ed., Method 972.41).
  • TLC Method I (AOAC OMA, current ed., Method 980.28).
  • TLC Method II- with potential interference material (AOAC OMA, current ed., Method 973.64).

Filth Exhibits: Fecal Material

  • Alkaline Phosphatase Test for Mammalian Feces (AOAC OMA, current ed., Method 981.22).
  • Alkaline Phosphatase Detection Method for Mammalian Feces in Corn Meal (AOAC OMA, current ed., Method 986.28 - See 990.10).
  • Alkaline Phosphatase Detection Method for Mammalian Feces in Grain (AOAC OMA, current ed., Method 990.10).
  • Alkaline Phosphatase Detection Method for Mammalian Feces in Ground Black Pepper (AOAC OMA, current ed., Method 993.27).
  • TLC Coprostanol for Mammalian Feces (AOAC OMA, current ed., Method 988.17).
  • Microchemical test for Uric Acid (AOAC OMA, current ed., Method 962.20).
  • TLC Method for Uric Acid (AOAC OMA, current ed., Method 986.29).
  • Spectrophotometric Method for Uric Acid (in Flour) (AOAC OMA, current ed., Method 969.46).

References for bone and skull confirmation:

  1. Popesko, P., Rajtova, V. and Horak, J., (1990). A color atlas of the anatomy of small laboratory animals (Vol. 1, Rabbit, Guinea Pig; Translated 1992, ISBN 0 7234 1822 5). London: Wolfe Publishing, Ltd.
  2. Popesko, P., Rajtova, V. and Horak, J.,(1990). A color atlas of the anatomy of small laboratory animals (Vol. 2, Rat, Mouse, Hamster; Translated 1992, ISBN 0 7234 1823 3). London: Wolfe Publishing, Ltd.
  3. Gottschang, J. L. (1981).  A guide to the mammals of Ohio (ISBN 0-8142-0242-X).  Columbus, OH: Ohio State University Press.
  4. Nowak, R. M. (Ed.). (1991). Walker's mammals of the world (5th ed.). Baltimore: John Hopkins University Press. 
  5. Hamilton, W. J., Jr., Whitaker, J. O., Jr. (1979). Mammals of the eastern United States (2nd ed., ISBN 0-8014-1254-4, LC 79-12920). New York: Cornell University Press.

References for Forensic Entomology:

  1. Smith, K. G. V. (1986). A manual of forensic entomology. London: British Museum (Natural History) and Ithaca, New York: Cornell University Press.
  2. Hall, R. D. and  Haskell, N. H., Wecht, C., (Ed. Forensic Sciences). (1995). Forensic entomology; applications in medicolegal investigations. New York:  Mathew Bender.
  3. Byrd, J. H. and Castner, J. L. (2001). Forensic entomology; the utility of arthropods in legal investigations. Boca Raton, FL: CRC Press

 4.4.6 Specialized Microscopy Techniques, Optical Crystallography (a.k.a. Polarized Light Microscopy), and ID Spot Tests

A. Objectives

Microscope discussions in an earlier section (Section 4.2.2), were intended only to introduce the variety, principles, set-up and maintenance of microscopes. It was not intended to teach the practical application of those tools. This chapter shows specialized microscopy methods and techniques, in particular those used in optical crystallography, as they apply to common problems often encountered in the filth laboratory.

B. Discussion

A thorough discussion of the principles of optical crystallography is beyond the scope of this manual, and in situations requiring advanced crystallographic techniques, e.g. measurement of more than one refractive index, measured structure, or precise optical characterization, these topics will be handled as a specialized course. An excellent discussion of this topic appears in the Food and Drug Administration Bulletin No. 1, Chapter X, reprinted in FDA By-Lines Vol. 6(1):20-53 (July 1975) and the instructor will identify the parts that should read be read. It will give the trainee more insight into the science of crystallography and the need for advanced study. This chapter will be discussing particular problem analytes and how they are analyzed in the lab, through polarized light microscopy or phase contrast microscopy, and the use of "spot testing" or micro-chemical tests to confirm visual findings. 

In the past, the main thrust of the science of optical crystallography or PLM has been the identification of crystalline drug substances. Even today, in the hands of a skilled analyst, PLM provides a rapid and accurate identification of many substances and avoids elaborate and costly chemical analyses. But the work can be tricky; after learning, practice frequently for reliable results. 

PLM has had a long history in FDA. The earlier work provided a reference catalog of known optical properties of crystalline substances. Most district laboratories have this index card catalog covering the thousands of substances (mostly drugs) studied. However, substances falling within the framework of filth analysis include glass, struvite, urea, dixanthylurea and the starches. Precise identification of these substances, and perhaps others, is most easily accomplished by optical crystallography, and/or with phase contrast microscopy; many are spot tests in and of themselves, or spot tests can be used as confirmation of observations.

C. Assignment

Under the direction of an experienced analyst, study the principles, methods, and techniques of optical crystallography found in the Food and Drug Technical Bulletin No. 1, 2nd Ed. 1981. (Note: The trainee is to have the opportunity for first-hand observation of the optical phenomena described in that publication.)

 Learn to apply the microscope and spot testing to the identification of the following substances: 

  • glass
  • struvite
  • urea and dixanthylurea
  • corn, wheat, potato, soy, and rice starch

D. Evaluation

  1. Demonstrate an understanding of the following optical phenomena: Becke lines; birefringence; crystal habit; extinction; isotropism and anisotropism; Newton's rings; cross polarization; refractive index 
  2. Using an unknown crystalline substance provided by the trainer, correctly determine the refractive index of an isotropic substance.
  3. Identify the unknown substance given by the instructor (#2 above) making additional observations.