Camacho, A., M.Giles, A.Ortegn, M.Palao, B.Serrano and O.Velzquez. 2009.

Techniques for Analysis

Microbiological Food. 2nd ed. Faculty of Chemistry, UNAM. Mexico.

Method for the account of molds and yeasts in foods.

OBJECTIVES

Perform properly by plate count method, the number of viable yeasts and molds in foodstuffs intended for
human consumption.

To evaluate the microbiological quality of a food, it is feasible to be contaminated by these organisms, with
reference NOM-111SSA1-1994.

Understand the basis for all steps of the method of detection and quantification of molds and yeasts in food.

GENERAL

Fungi and yeasts are widely distributed in the environment, they can be found as normal flora of a food or as
contaminants in poorly sanitized equipment. Certain species of fungi and yeasts are useful in preparing some
foods, but can also be caused the decomposition of other foods. Because of its slow growth and low
competitiveness, fungi and yeasts are manifested in foods where bacterial growth is less favorable. These
conditions may be low pH, low humidity, high salt or carbohydrate, low temperature storage, the presence of
antibiotics, food or exposure to irradiation. Therefore they can be a potential problem in fermented dairy foods,
fruits, fruit drinks, spices,

Fungi and yeasts can use certain substrates such as pectins, carbohydrates such as polysaccharides, organic
acids, proteins and lipids. They can also cause problems by: (a) synthesis of (mycotoxins) toxic metabolites, (b)
resistance to heat, freezing, antibiotics or radiation and (c) ability to alter unfavorable substrates allowing the
growth of pathogenic bacteria. They can also cause odors and flavors and discoloration of the food surfaces.

The term mold It is usually applied to designate certain multicellular filamentous fungi whose growth on the
surface of food is often easily recognized by its velvety or cottony, sometimes pigmented. Usually all moldy food
is considered unfit for consumption. Identification and classification of molds is based on macroscopic and
microscopic observations.

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Microbiological Food. 2nd ed. Faculty of Chemistry, UNAM. Mexico.

Hyphae and mycelium. The thallus of molds consists of a mass of branching filaments called hyphae mycelium
calling to all the hyphae. Hyphae can be submerged or air. They can also be classified into vegetative hyphae or
growing, which are responsible for nutrition mold, and fertile hyphae or hyphae that produce reproductive
organs. Molds are divided into two groups: septate, ie provided with transverse partitions dividing the hyphae,
and no septate cenocticos whose hyphae are formed by cylinders without transverse partitions. This type of
hyphae has scattered along the cylinder and are called multicellular nuclei. Mycelial certain structures or certain
organs help identify molds. For example rhizoids or "anchors" gender Rhizopus or Absidia, the basal cell of the
genus Aspergillus and dichotomous branching, or Y-shaped, gender Geotrichum.

Reproductive organs or structures. Molds mainly reproduce by asexual spores. Some molds also produce
sexual spores. In such fungi they are called "perfect", which are divided into Oomycetes Y

Zygomycetes if not septate, or in Ascomycetes Y Basidiomycetes if they are septate, as opposed to the
"imperfect" molds, Fungi imperfecti, which they have only asexual spores.

asexual spores. Molds produce many asexual spores are small, lightweight and resistant to desiccation. They
are easily spread by the atmosphere and lead to pellet the thallus of a new mold. The three main types of
asexual spores are: (1) conidia, (2) artrosporas or mildews and (3) sporangiospores. The conidia are separated
or grow under certain fertile hyphae called conidiophores and are generally free, ie, not within any receptacle,
as opposed to sporangiospores, which are within a sporangium or receptacle, located in the end of a fertile
hyphae, the sporangiophore. The artrosporas are formed by fragmentation of hyphae. The thickened end of the
sporangiophore is called columella and adopts typical of each species of mold shapes.

physiological properties. Compared to most yeasts and bacteria, the most molds require less moisture supply.
A total moisture content below 14 to 15 percent flour or some nuts prevent or retard much the growth of molds.
Molds could be considered mesophilic, ie they are able to grow well at normal temperatures. The optimum
temperature for most are around 25 to 30 C, although some are psychrotrophic and some are thermophilic.
They are aerobic, this is true at least in molds that grow on the surface of food. Almost all molds are able to
grow over a wide pH range (pH between 2 and 8.5), although most grows best at acidic pH. They are able to
use many types of food, ranging from simple to complex. They possess hydrolytic enzymes, and hence some
are used for industrial production of amylases, pectinases, proteases and lipases.

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Microbiological Food. 2nd ed. Faculty of Chemistry, UNAM. Mexico.

Some genres molds important food.

Mucor. Involved in altering some foods and are used in the manufacture of others. M. rouxii It is used for the
saccharification of starch for cheese ripening and for the manufacture of some oriental foods.

Rhizopus. the species R. stolonifer, or bread mold is very common and is involved in altering certain foods:
berries, fruits, vegetables, bread, etc.

Aspergillus. The aspergilli are abundant molds. Some species involved in alterations experiencing food, while
others are useful for preparing certain foods. A. niger It is used for industrial production of citric and gluconic acid
and certain enzymes. A. flavus It is used for the manufacture of certain oriental foods and obtaining enzymes.

They have been reported almost 50 species Aspergillus as producers of toxic metabolites generally called
mycotoxins. The most important are: called aflatoxins (produced by A. flavus, A. parasiticus Y A. nomius); ochratoxin
A produced by A. ochraceus A. carbonarius Y A. niger; produced mainly sterigmatocistina by A. versicolor; cyclopiazonic
acid produced by A. flavus Y A. tamarii. Citrinin, patulin and peniclico acid are also produced by species of Aspergillus,
the tremorgnicas toxins are produced by A. terreus

(Territremas), A. fumigatus ( fumitremorgenas) and A. clavatus ( triptoquivalina).

Aflatoxins are derived difurancumarina. Aflatoxin B 1, B 2, G 1 and G 2 They are produced in nature by molds already
mentioned. Points B and G refer to colors flurescencia by name in English (blue and green, respectively)
observed under wavelengths in the UV region, and the subscripts 1 and 2 relate to chromatographic separation
patterns. Aflatoxins M 1 and M 2 They produced by hydroxylation of the respective aflatoxins B. Aflatoxin B 1 perhaps
the most powerful carcinogenic animal including human liver. Ochratoxin A and citrinin affect kidney function.
The tremorgnicas toxins affect the central nervous system.

Penicillium. It is another kind of molds frequent occurrence and importance in food, P. expansum It produces soft
rot of fruits; P. digitatum Y P italicum produce citrus rot. The species P. camemberti, P. roqueforti They used in
cheese ripening.

They have reported more than 80 species Penicillium as producers of mycotoxins. These mycotoxins can be
divided into two groups: those that affect the function of the liver or kidney, and neurotoxins. The main
mycotoxins produced by
Penicillium are, ochratoxin A, citrinin, patulin, cyclopiazonic acid, citreoviridin, penitremo A, PR toxin and
roquefortina and secalnico acid. Of these ochratoxin A it is undoubtedly the most important, is a renal
carcinogen and is produced by P. citrinum, P. viridicatum, P. verrucosum. The main source of this toxin is rye
bread or

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Microbiological Food. 2nd ed. Faculty of Chemistry, UNAM. Mexico.

wheat, or through these grain fed pigs whose meat is then consumed by humans.

It is recommended to consult the literature to meet other genera of molds with importance in food.

Yeast and yeast-like fungi.

He term yeast refers to those fungi that are not usually filamentary, but unicellular and ovoid or spheroid, and
reproduce by budding or fission.

Yeast found in foods can be beneficial or harmful. Yeasts are used in food processing such as bread, beer,
wine, vinegar and cheeses are also used in obtaining enzymes and fermented foods. Yeasts are harmful when
they produce the alteration of sauerkraut, fruit juices, syrups, molasses, honey, meats, wine, beer and other
foods.

Morphological characteristics of yeast are determined by microscopic observation. Its shape can be spherical to
ovoid from, lemony, pyriform, cylindrical, triangular and even elongated. Most reproduce asexually by budding
or multicellular polar budding. A few species reproduce by fission.

In the cultures on agar plates it is difficult to distinguish yeast colonies of the bacterial colonies; microscopic
observation of microorganisms is the only sure way to differentiate. Most young yeast colonies are wet and
some mucosa; Most colonies are whitish, although some have a cream or pink. They are oxidative, fermentative
or metabolic activity is at once both.

Most yeasts grow better with a high moisture content. However, growing better than most bacteria on substrates
containing high concentrations of solutes (eg carbohydrates or sodium chloride), ie are osmotolerant. However
most of the yeasts need more moisture than molds. For most of the yeast TO w minimum growth ranges between
0.88 and 0.94. The growth temperature range is similar to the mold, with an optimum temperature around 25 to
30 C and a maximum temperature around 35 to 47 C. They grow best aerobically, although the type
fermentative species are able to grow, albeit slowly, in anaerobiosis. Sugars are the most appropriate energy
source for the yeast, although oxidative, can oxidize organic acids and alcohol.

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Microbiological Food. 2nd ed. Faculty of Chemistry, UNAM. Mexico.

Some kinds of food are important:

Schizosaccharomyces. Yeasts of this genus are found in tropical fruit in molasses and honey.

Saccharomyces. the species S. cerevisiae is used in many food industries, as in the leavening of bread,
beer fermentation,
fermentation of wines, in the production of alcohol, glycerol and invertase.

Kluyveromyces. K. marxianus ( before Saccharomyces fragilis) It used in the

production of dairy products for their ability to ferment lactose.

Zygosaccharomyces. Yeasts of this kind are important for their ability to grow in media with high concentrations
of sugar (osmophilic), involved in altering honey, syrups and molasses, and also in the fermentation of soy
sauce and some wines.

Pichia. Growing on the surface of the liquid forming a film. P. membranafaciens It produces a film on the surface
of the beers and wines.

Debaromyces. Forman film on the surface of the brines. D. kloeckeri It grows on the surface of cheeses and
sausages.

Hanseniaspora. These yeasts are lemon shaped and grow fruit juices.

Torulopsis. Cause problems in breweries. T. sphaerica ferment lactose, altering dairy products. Other species
alter sweetened condensed milk, fruit juice concentrates and acidic foods.

Candida. the species C. utilis It is grown for the production of single cell protein to incorporate both food for human
consumption and animal feed. the species C. krusei used in conjunction with starter cultures for dairy products. C.
lipolytica alteration produces butter and margarine.

Brettanomyces. Produce large amounts of acid and are involved in the fermentation of beer Belgian "lambic"
type, ales and French wines.

Trichosporon. These yeasts grow best at low temperatures, being in the beer factory and on the surface of
chilled beef.

Rhodotorula. These yeasts red, pink or yellow, can cause spots on the surface of the food as the surface of the
meat, or pink areas in sauerkraut.

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 Camacho, A., M.Giles, A.Ortegn, M.Palao, B.Serrano and O.Velzquez. 2009. Techniques for Analysis
Microbiological Food. 2nd ed. Faculty of Chemistry, UNAM. Mexico.

BASIS

The method is based on inoculating a known amount of sample in a selective culture medium specific, using the
capabilities of this microbial group to be used as nutrients polysaccharides containing medium. Hydrolysis of
these compounds is effected by enzymes having these microorganisms. The survival of fungi and yeasts at pH
acids is revealed to inoculate the culture medium acidified to a pH of 3.5. Also, acidification allows the
elimination of most bacteria. Finally, aerobic conditions and incubation at 25 1 C results colony growth
characteristics for such microorganisms.

CULTURE MEDIA AND DILUYENTES

1 Erlenmeyer flask 250 ml capacity, containing 90.0 mL of phosphate buffer solution pH 7.2 or 0.1%
peptone water, sterile to.
3 tubes of 16 x 150 mm, containing 9.0 mL of phosphate buffer solution pH 7.2 or 0.1% peptone water, with
sterile cotton plug to.
4 22 x 175 mm tubes with 20.0 mL of potato dextrose agar to .
4 22 x 175 mm tubes with 20.0 mL of malt extract agar to.

NOTE

Mexican Official Standard uses the potato dextrose agar only for detection and quantification of these groups of
microorganisms. However it is suggested for quantification of yeasts, the use of malt extract agar acidified as it
is richer in nutrients and suitable for the development of this microbial group means.

SOLUTIONS, SOLUTIONS AND INDICATORS

Lactophenol blue dye solution cotton b.

Gram staining dyes b.
Sterile solution of tartaric acid at 10.0% to.

MATERIALS AND EQUIPMENT

Sterile glass blender or stomacher bag to.

Motor for blender or Stomacher to.
Sterile pipettes of 1 mL with cotton plug to.

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Microbiological Food. 2nd ed. Faculty of Chemistry, UNAM. Mexico.

Sterile pipettes 10 mL with cotton plug to.

Sterile Pasteur pipettes a, b
propipette a, b
Sterile Petri boxes (one is used for weighing the sample) to.
utensils sterile sample handling knives, spoons, forks, etc. to

Incubator thermostat that can be maintained at 25 1 C to.

Water bath with temperature control and mechanical circulation maintained at 45 1 C to.

Slides, cover slips, diurex b.

Optical microscope b.
bacteriological loop and handle mycological b.

Colony counter darkfield, with adequate lighting, plate glass magnifying lens gridded b.

NOTES

to
Material needed for the start of practice.
b
Equipment necessary for 3, 4 and / or 5 days after the start of practice.

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 DETERMINATION
agar
Version Manager Manuals and Documents (AmyD). Faculty of Chemistry, and
UNAM / or malt extract agar acidified with 0.3 mL 10% tartaric acid cooled to 45 C on each plate

Homogenize

with 90

mL of solution 0.0
Incubate

/ 3,4 or 5 days

Fold dilutions using 9.0 ml tub

28 C
1.0 mL

10- 1

plates upside

eachDuplicate deposit

10- 2 1.0 mL
dilution in sterile Petri dishes A., M.Giles, A.Ortegn,

Counting
those
plates with between 10 to 150 colonies of fungi and / or
1.0 mL
yeast
10- 3

1.0 mL
as
indicating

CFU / g
Y

report
weather
or 10- 4

8 mL
separately
incubation
shows,
 Camacho, A., M.Giles, A.Ortegn, M.Palao, B.Serrano and O.Velzquez. 2009. Techniques
Microbiological analysis of food. 2nd ed. Faculty of Chemistry, UNAM. Mexico.

PROCESS

1. Weigh 10.0 g of sample in a sterile Petri dish and pass to an Erlenmeyer flask containing 90.0 mL of
a phosphate buffer solution pH 7.2 or 0.1% peptone water.

2. Homogenize the sample with the above solution in a glass of sterile blender or pass to a Stomacher
bag and homogenize for 10.0 sec for the blender at low speed or in the Stomacher 30.0 sec at a
normal speed. This is the primary dilution.

3. suspension or above solution, 1.0 mL taken and transferred to a test tube containing 9.0 mL of
phosphate buffer solution pH 7.2, stirring and repeating this operation as many times as necessary
dilutions. You should use a sterile pipette for each dilution.

NOTE

Filamentous microorganisms form colonies from a spore or a hypha or fragment of a mass of hyphae.
Because of this, the number of CFU / mL may vary depending on the conditions of homogenisation of
the sample (the longer the greater homogenization will rupture hyphae and therefore the CFU / mL
would be increased), so that particular care should be at this stage. Homogenization times cited in this
methodology are recommended in the Official Mexican Standard for this microbial group.

4. Place duplicate in sterile Petri boxes, 1.0 mL of each sample dilutions, using a sterile pipette.

5. Melt the medium contained in the pipes 22 x 175 mm with 20.0 mL of agar potato dextrose and / or
sterile malt extract agar. Cool them and keep them 45 C.

6. To achieve acidify media at pH 3.5, added per 100.0 mL of agar, 1.4 mL of 10% tartaric acid sterilized
by membrane filtration, or the solution sterilized at 121 C 1 C for 15 minutes. This means each
containing 20.0 mL of medium and kept molten tube 45 C was added 0.3 mL should acid, or
place in the Petri dish being careful not to touch the sample before adding culture medium.

7. After acidification, using a tube acidic medium as control and measure the pH to corroborate that is at
3.5 using a potentiometer.

8. In each Petri dish with the inoculum, pour 15.0 20.0 potato dextrose agar mL acidified and / or malt
extract agar acidified, melted and kept at Four. Five C. The time between the preparation of the
dilutions and when being poured the culture medium must not exceed 20.0 min.

9. Gently mix the medium with six moves from right to left, six in the direction of clockwise,
counterclockwise six six back and forth, on a smooth surface, having

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Microbiological analysis of food. 2nd ed. Faculty of Chemistry, UNAM. Mexico.

careful not to moisten with half the top of the petri dish. Allowing the mixture in the Petri plates to
solidify, allowing them to stand on a cold horizontal surface.

10. Check the sterility of the media for which acidified is poured into a Petri dish without inoculum 15.0
20.0 potato dextrose agar mL of acidified and / or malt extract agar acidified. After incubation these
boxes must not present colony development.

11. Invert the boxes and placed in the incubator at 25 1 C.

12. Count colonies from each plate after 3, 4 and 5 days incubation. After 5 days, select those dishes
containing between 10 and 150 colonies. If any of the boxes shows widespread mold growth or if it
is difficult to have well-isolated colonies, consider
the
Quantification 4 days incubation or even 3 days. In this case, the incubation period reported on the
results of the analyzes.
13. Perform a wet mold staining blue dye cotton Lactophenol for microscopic examination and possible
identification molds they have been developed.

14. Perform a Gram stain for microscopic observation of the obtained yeast.

15. Count colonies each representative plaque after 3, 4 and 5 days of incubation (at 26 1 C or room
temperature).
16. Consider accounts plates 10 to 150 colonies as appropriate for the report. Multiplied by the inverse
of the dilution.
17. Report colony forming units per gram or milliliter (CFU / g
mL) of molds and yeasts (each independently), incubated at 25 1 C for 5 days.

18. To describe the macroscopic and microscopic characteristics observed, the molds and / or yeasts
developed from the sample.
19. If any part of the box shows widespread fungal growth, or if it is difficult to count the colonies,
consider the development of these microorganisms after 4 days incubation and even after 3 days.
In this case you must inform the incubation period of 3 or 4 days, in the analysis results.

CALCULATION AND EXPRESSION OF RESULTS

To report results suggested follow the instructions below are expressed:

QUANTITATIVE ANALYSIS

To make a report on the quantitative part of this analysis should be selected

plates containing between 10 and 150 colonies
(Statistically representative). Later, count the colonies present and calculate the number of fungi and
yeasts separately. Thus one can calculate the colony forming units per gram or per milliliter depending
on the physical state of the sample. This is achieved

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Microbiological analysis of food. 2nd ed. Faculty of Chemistry, UNAM. Mexico.

multiplying the number of CFU found in a representative case by the reciprocal of the dilution
corresponding to that box.

In the case of plates containing less than 10 colonies (CFU) of fungi and / or yeasts, it should report the
number of CFU obtained indicating the corresponding dilution.

In the case of not finding characteristic colonies fungi and / or yeasts to report the result is less than 10
CFU / g or less well than 10 CFU / mL (sensitivity method)

For any of the above cases should report incubation time in which quantification was performed.

For any of the above cases should report separately the result of quantifying fungi and yeast.

Report macroscopic and microscopic of different types of colonies of molds and yeasts observations
obtained during analysis. Report if possible or probable genres.

BIBLIOGRAPHY.

Norma Oficial Mexicana. NOM-111-SSA1-1994. Goods and services. Method for the account of
molds and yeasts in foods.

W. & D. Frazier Westhoff (1994) Food Microbiology. 4th. ed. Acribia, Spain. 23-50.

Beuchat LR & Cousin MA (2001) "Yeasts and Molds". In: Compendium of Methods for the
Microbiological Examination of Foods. 4 th ed. Downs FP & Ito
K. (Eds.) APHA. Washington. 209-215.

M. L. Pierson & Smoot (2001) Indicator Microorganisms and Microbiological Criteria. In: Food
Microbiology. Fundamentals and Frontiers. 2 nd ed. M. L. Doyle Beuchat & Montville T. (Eds.) ASM Press.
USES. 71-87.

Hocking A. (2001) toxigenic Aspergillus Species. In: Food Microbiology. Fundamentals and Frontiers.
2 nd ed. M. L. Doyle Beuchat & Montville T. (Eds.) ASM Press. USES. 451-465.

Pitt J. (2001) toxigenic Penicillium Species. In: Food Microbiology.

Fundamentals and Frontiers. 2 nd ed. M. L. Doyle Beuchat & Montville T. (Eds.) ASM Press. USES.
467-480.

Food and Drug Administration (2003) " Bacteriological Analytical Manual ". 9 th
ed. Arlington, VA: AOAC.
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Microbiological analysis of food. 2nd ed. Faculty of Chemistry, UNAM. Mexico.

International Commission on Microbiological. Specifications of Foods (2005) " Microorganisms in

Foods 6 " Chapman & Hall. 2nd ed.

http://www.cfsan.fda.gov/~ebam/bam-1.htmL

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