Government of Canada Gouvernement du Canada

HPB Method MFHPB-02

October 2006

HEALTH PRODUCTS AND FOOD BRANCH

OTTAWA

METHOD FOR THE DIRECT MICROSCOPIC EXAMINATION OF FOODS

IN HERMETICALLY SEALED CONTAINERS

Alexander Fabricius Patti Wilson

Fish/Food Laboratory CFIA Dartmouth Laboratory
Canadian Food Inspection Agency 1992 Agency Dr
Mississauga, Ontario, L5T 2R4 Dartmouth, NS, B3B 1Y9

E-mail: fabriciusa@inspection.gc.ca E-mail: wilsonpa@inspection.gc.ca

Microbiological Methods Committee Dev C. Nundy

Health Products and Food Branch Food Laboratory
Microbiology Evaluation Division Laboratory Services Division
Bureau of Microbial Hazards, Food Directorate Canadian Food Inspection Agency
Postal Locator 2204A1 Ottawa, Ontario, K1A 0C6
Ottawa, Ontario, K1A 0L2
E-mail: dnundy@inspection.gc.ca
E-mail: Don_Warburton@hc-sc.gc.ca

1. APPLICATION

This method can be used for the microscopic examination of incipient or non-incipient type spoiled foods in
thermally processed hermetically sealed containers, that are suspected of being compromised
microbiologically . Ascertaining morphological characteristics of organisms is necessary for the purposes of
determining whether it has public health significance or not. This revised method replaces MFHPB-25G,
dated October 1991.

2. PRINCIPLE

The method describes the microbiologically suspect contaminated canned foods or other hermetically sealed
containers that may be used in the preparation of simple stained smears and wet mounts, their microscopic
examination and recording of results.

NOTE: If the analyst uses any variations of the staining procedures listed here (either product that is
commercially available or made from scratch), it is the responsibility of the analyst or Laboratory
Supervisor to ensure equivalency.
3. DEFINITION OF TERMS

Published on the Food Directorate’s (Health Canada's) website at

http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index_e.html
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See Appendix A of Volume 2 for definitions not covered in this section.

3.1 Hermetically Sealed Containers: These are containers designed to be secure against the entry of air
and microorganisms into the food product. These containers may be: 1) rigid, such as glass bottles
and two or three piece metal cans; 2) semi-rigid, such as disposable serving dishes used to contain
some ready-to-eat meals, and 3) flexible, such as retort pouches.

3.2 Semi-Liquid Products: These are products that may pour with difficulty, but can be mixed by shaking
the container.

3.3 Semi-Solid Products: These are products that have a high viscosity; they pour with great difficulty,
and cannot be mixed by shaking the unopened container.

3.4 Acid and Acidified Food: a food with a pH equal to or lower than 4.6.

4. COLLECTION OF SAMPLES

4.1 See Appendix B of Volume 2 and follow instructions, if applicable. Depending on the circumstance
(container type, food type and the number of samples requiring microscopic examination), containers
may be examined individually.

5. MATERIALS AND SPECIAL EQUIPMENT

1) All materials and special equipment specific to the aseptic analysis of foods in hermetically sealed
containers, as listed in MFHPB-01

2) Sterile bacteriological sampling loops, applicator stick, transfer pipette or other appropriate utensil for
obtaining a smear sample

3) Light microscope with a 100 x oil immersion objective

4) Microbiological dyes, such as ethanol based solutions of crystal violet or methylene blue

5) Xylol

6) Clean microscope slides and cover slips

7) 0.05N Sodium hydroxide (NaOH) solution

6. SAFETY PRECAUTIONS

HANDLE ALL SWOLLEN CONTAINERS AS IF THEY CONTAINED CLOSTRIDIUM BOTULINUM AND/OR

THEIR TOXINS UNTIL THE ABSENCE OF SUCH HAS BEEN ESTABLISHED.

NEVER TASTE FOOD FROM A CAN UNDER INVESTIGATION. ALWAYS USE MECHANICAL PIPETTING
DEVICES.

SWOLLEN CONTAINERS, ESPECIALLY HARD SWELLS, CAN SPRAY THEIR CONTENTS UPON BEING
PUNCTURED. TO PREVENT CONTAMINATION OF SURROUNDING ENVIRONMENT, SUITABLE
HANDLING PRACTICES MUST BE USED. IT IS RECOMMENDED THAT SWOLLEN CONTAINERS BE
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HANDLED IN A CLASS II BIOSAFETY CABINET WHICH PROVIDES PROTECTION TO THE OPERATOR

AS WELL AS THE PRODUCT.

NEVER FLAME HARD SWELLS SINCE THE ABSORBED HEAT MAY INDUCE THE CANS TO BURST.

DYES AND SOLVENTS CAN BE HARMFUL. REVIEW MATERIAL SAFETY DATA SHEETS AND USE
PERSONAL PROTECTIVE EQUIPMENT AS APPROPRIATE.

7. PROCEDURE

7.1 Handling and Preparation of sample units

7.1.1 Follow MFHPB-01, for the identification of the container, removal and identification of the
label, aseptic opening of the container, and mixing the contents (as applicable).

7.2 Preparation of a dry smear

Try, where possible, to include some solid particles in the sample. Smear the sample evenly in a
thin layer on the surface of the slide. The area of the smear should be about 2x3 cm. Break solid
lumps with the loop during smearing as smears that are too thick may prevent adequate light
penetration for microscopic examination. Air dry. The smear is then fixed by gently passing the
slide several times through a burner flame. Avoid over heating since this may result in cell
distortion and/or breakage of the slide.

7.2.1 Liquid and semi-liquid foods

Mix contents thoroughly. Using a sterile bacteriological sampling loop, take a sample of
the contents and transfer it to a clean glass microscope slide. Spread to form a thin film.

7.2.2 Solid and semi-solid foods

Microorganisms may be localized in solid and semi-solid products. They are more likely
to be in the centre of the product if under processing has occurred, or near the seams or
closure if the container has leaked.

With this in mind, using an appropriate sterile tool, scrape the external product surface,
especially the area in close proximity to the seams and/or aseptically obtain a smear
sample from the interior of the product, particularly from the centre core. A drop of distilled
water may be required to permit distribution of the material on the surface of the slide into
a thin film.
7.2.3 High fat foods and foods prepared in oil

Foods canned in oil or having a high fat content are difficult to stain. Smears from such
foods should be carefully drained if possible and/or defatted by immersing the heat fixed
slides in xylol for 1-2 minutes. Remove and allow to dry before fixing.

7.3 Staining of the dry smear

When examination of a smear for morphological types and cell concentration is required, a simple
stain (using a single dye) such as crystal violet or (Loeffler) methylene blue applied to a dry, fixed
smear is adequate. Gram staining is not recommended for direct smears because the presence of
old or stressed cells and over decolourisation can lead to erroneous Gram reactions.
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7.3.1 Simple stains

Crystal violet - Apply crystal violet solution to the fixed smear; leave for 10 seconds;
gently rinse with distilled or tap water and blot dry.

Methylene Blue - Follow instructions as stated for crystal violet, except leave stain on slide
for 30 seconds.

7.3.2 Smears prepared from foods packed in syrups or brine

After staining, remove excess stain by dipping the slide gently in a beaker of distilled
water. Do not hold it under a stream of running water. Allow to air dry and do not blot.

7.3.3 Acid foods

Acidity sometimes causes excessive precipitation of the stain on the slide making the
smear difficult to examine. This can be prevented by neutralisation of the acidity as
follows: add one loopful of sample to a clean glass slide and mix with a drop of 0.05N
NaOH. (For example, if you add one drop of 0.05N Na0H to one (10 :l) loopful of food
having a pH of 3.5, it will give a result of approximately pH 7.00). Follow MFHPB-03 to
determine the pH of foods.

7.4 Examination of stained smears

Microscopically examine the stained smear using an oil immersion 100 X objective. At least one-
half of the smear should be scanned to locate areas with the best identifiable cell concentrations
and variety of morphological types. At least five of these fields should be examined. Count the
different morphological types identified in each field separately.

7.5 Recording Results

See Appendix A of this method for a table which can be used for recording results.

7.5.1 Note the general bacterial morphological types and determine the approximate numbers
of these types present in the five fields. Report the average count per field for each
morphological bacterial type.

7.5.2 If no cells are observed when scanning the slide, report that no cells were detected.

8. REFERENCES

8.1 American Public Health Association (APHA). 2001. Compendium of Methods for the
Microbiological Examination of Foods. Fourth edition. F.P. Downes and K. Ito (editors), American
Public Health Association, Washington, D.C.

8.2 American Public Health Association (APHA). 1992. Standard Methods for the Examination of
Dairy Products. Chapter 6. Sixteenth edition, American Public Health Association, Washington,
D.C.

8.3 W. L. Landry, A. H. Schwab, and G. A. Lancette. 1995. Examination of Canned Foods In:
Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. Chapter 21A. Published by AOAC
International, Gaithersburg. MD.
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Website: http://www.cfsan.fda.gov/~ebam/bam-toc.html

8.4 National Food Processors Association. 1980. Laboratory Manual for Food Canners and
Processors, Vol. 1, National Food Processors Association. AVI Publishing Company Inc.,
Westport, Connecticut.
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October 2006

APPENDIX A

STAIN USED: MICROSCOPIC EXAMINATION

Field No.
Sample ID Average
1 2 3 4 5

Morphological Count Morphological Count Morphological Count Morphological Count Morphological Count
type type type type type