PROTOCOLS

MICROBIOLOGICAL
ANALYSIS IN
PHARMACEUTICAL
INDUSTRY
Inspired by knowledge
ALL PROCEDURES ACCORDING TO EUROPEAN PHARMACOPEIA
Table of Contents

MICROBIOLOGICAL WATER TESTING________________ 04

Ph. Eur. 9.0

STERILITY TESTING________________________________ 05
Procedure according to Ph. Eur. 2.6.1

NON-STERILE PRODUCT TESTING

Procedure according to Ph. Eur. 2.6.12

TOTAL MESOPHILIC ENUMERATION____________________ 07

ESCHERICHIA COLI__________________________________ 09

BILE-TOLERANT GRAM-NEGATIVE BACTERIA____________ 10

SALMONELLA______________________________________ 11

STAPHYLOCOCCUS AUREUS__________________________ 12

PSEUDOMONAS AERUGINOSA_______________________ 13

CLOSTRIDIUM______________________________________ 14

CANDIDA__________________________________________ 15

2
The pharmaceutical industry is one of the world's most
influential business sectors, comprised of public and
private organizations dedicated exclusively to the
research, manufacture, marketing and distribution of
pharmaceutical chemicals for human and animal health.
Regulating the analysis and control of medications in this
sector is extremely important both in the development
of new drugs and in the control of existing products in
order to ensure their quality, effectiveness and safety.
The European Pharmacopoeia is the official legal
instrument in the EU established to ensure the quality of
this type of products. It includes quality standards for
active principles, general standards for formulation, and
general standards for manufacturing medications. It is
made up by Expert Committees of the countries involved
and other countries, as well as the WHO, participate as
observers.
This standard considers the microbial analysis of
pharmaceutical products, raw materials used, water,
air, equipment or packaging material, among others,
which are vital to minimize the type and number of
microorganisms present. Those products of parenteral
administration or for use in eyes must be sterile.
Drugs are considered contaminated when it exceeds
a limit of opportunistic or objectionable pathogenic
microorganisms, which have the capacity to limit the
effectiveness of the product, when they present toxic
metabolites or physical or chemical deterioration.
The ineffective dose varies between species and even
between individuals.

This guide will describe the culture media and

the procedures formulated according to the
Pharmacopoeia required to perform:
• Sterility testing
• Non-sterile product testing
• Microbiological count testing
• Microbiological water testing

3
Microbiological
Water Testing

Water is the most commonly used substance in the

pharmaceutical industry, both for production, as an
ingredient in the product or in one of the manufacturing
stages; and for washing equipment. Microbiological
control of water is of vital importance at entrance to the
treatment plant, purification, storage, and distribution
stages since microorganisms can proliferate throughout
any stage described above, affecting its quality.

Water samples should be analyzed to avoid changes in Water for pharmaceutical use can be classified
the microbial count process. Once the sample has been according to use and method of production.
collected, it must be stored at a temperature between
• Water for injections: Used as a vehicle (bulk injection
2 and 10ºC. The count must be performed within 8
water) or as a substances/preparations diluent for
hours and the analysis of coliform bacteria within 30
preparing medications to be administered parenterally.
hours. Sampling, reception and analysis records must
demonstrate that the established timelines are met. • Purified water: Used for preparing medications that
require sterility and apyrogenicity, except those
authorized.
• Water for preparation of extracts: For preparing
medication extracts.
• Highly purified water: Its purpose is the preparation of
Bibliography medicinal products, for which a high biological quality
is required (except those cases in which water is used
for injectables).
American Public Health Association (1985) Standard Method for the
Enumeration of Water and Wasterwater.European Pharmacopeia 9.3.

PURIFIED WATER: 100 CFU/ml

Determine by means of a filter membrane with a pore size not
HIGHLY PURIFIED WATER: 10 CFU/100 ml exceeding 0.45 µm. Culture in R2A Agar (CAT. 1071)
Use at least 200 ml of sample Incubation at 30-35 ºC // 5 days

WATER FOR INJECTIONS: 10 CFU/ 100 ml

Use at least 200 ml of sample

WATER FOR PREPARATION OF EXTRACTS: Determine by means of a filter membrane with a pore size not
exceeding 0.45 µm. Culture in Trypticasein Soy Agar (TSA)
100 CFU/100 ml
(CAT. 1068)
STORED PURIFIED WATER: Incubation at 30-35 ºC // 5 days

100 CFU/ 100 ml

4
Sterility Testing

Introduction
The purpose of the sterility test is to verify the absence
of contamination by microorganisms in products that,
according to pharmacopoeias, are required to be
sterile, whether they have been sterilized or prepared
aseptically. A satisfactory result indicates the absence of
contaminating microorganisms in the sample examined
under test conditions. To achieve such conditions, the
test environment must be adapted to the way in which
the sterility test is performed. These working conditions
are regularly monitored by means of an appropriate
sampling of the working area.

This test appeared for the first time in the British

Pharmacopoeia in 1932, and later in the United States Bibliography
Pharmacopoeia in 1936 for sterile solutions. Over the
years, constant changes have been made, which have Brewer. JAMA, 115. 1940. Vera. J. Bact. 47:59, 1944.
Pittman. J. Bact. 51:19, 1946.
led to the improvement of techniques for detecting
contamination in this type of products. European Pharmocopoeia 9.3.

Aerobic, anaerobes, and fungus growth promotion test

AEROBIC BACTERIA ANAEROBIC AEROBIC BACTERIA FUNGUS

(Staphylococcus aureus, BACTERIA (Bacillus subtilis) (Candida albicans,
Pseudomonas aeruginosa) (Clostridium sporogenes) Aspergillus brasiliensis)

Inoculate in containers separated by microorganism Inoculate in containers separated by microorganism Trypticasein

Thioglycolate Liquid Medium (CAT. 1508) with < 100 CFU of Soy Broth (TSB) (CAT. 1224) with < 100 CFU of each species
each species described above. described above.

Incubate bacteria for no more than 3 days, and fungi and yeasts for no more than 5 days.

5
Method Suitability Test

This test must be carried out simultaneously with the sterility testing of the examined product. It is carried out when a
sterility testing of a new product needs to be carried out, or when the experimental conditions of the test change.
Use exactly the same procedure selected in the Product Sterility Testing described below except for the following
modifications.

A MEMBRANE FILTRATION METHOD B DIRECT INOCULATION METHOD

After transferring the contents on the container(s) of After transferring the contents on container(s) of the
the products to be examined to the filter membrane, products to be examined to the culture medium,
inoculate a small amount of viable microorganisms inoculate a small amount of viable microorganisms
(<100 CFU) to the final sterile diluent amount used to (< 100 CFU).
rinse the filter.

In both cases, use the same microorganisms described in the test for the growth promotion of aerobic, anaerobes and fungus.
Carry out a growth promotion test as a positive control. Incubate containers for a period not exceeding 5 days.

Sterility testing of the product to be examined

A MEMBRANE FILTRATION METHOD B DIRECT INOCULATION METHOD

Product to be analyzed: Product to be analyzed:
• Aqueous solutions • Oily liquids
• Soluble solids • Semi-solids (creams and ointments)
• Oils and oily solutions • Suture thread and related products for use in animals
• Semi-solids (creams and ointments)

Incubate inoculated media for 14 days.

Observe during the incubation time and, once period has elapsed, observe the macroscopic evidence of microbial growth. If the
medium becomes too turbid to read the presence/absence of microbial growth, transfer 1 ml to a new medium, and incubate it for
not less than 4 days.

If there is no evidence of microbial growth, the product complies with the sterility testing.

6
Microbiological
I count test
TAMC (Total Aerobic Microbial
Count) &; TYMC (Total Yeast and
Mold Counts)
The tests described in this section allow quantitative
enumeration of mesophilic bacteria, total aerobic microbial
(TAMC) and fungus count, total combined yeasts and mold
count (TYMC), which can grow under aerobic conditions

Bibliography

Standard Methods for the Examination of Dairy Products.

11th Edition. APHA., Inc. New York, 1960.
European Pharmocopoeia 9.3.

A - MEMBRANE FILTRATION B - PLATE COUNT METHODS

B.1 POUR PLATE METHOD

SAMPLE PREPARATION SAMPLE PREPARATION

At a concentration of 1:10, prepare the sample in At a concentration of 1:10, prepare the sample in
Buffered Peptone Water (CAT. 1401) at pH 7.0 and Buffered Peptone Water (CAT. 1401) at pH 7.0 and
homogenize. homogenize.

This stock solution can be supplemented with neutralizersand This stock solution can be supplemented with neutralizers and
surfactants surfactants

MICROORGANISMS MOLDS AND MICROORGANISMS MOLDS AND

AEROBIC TOTALS YEASTS TOTAL AEROBIC TOTALS YEASTS TOTAL
Transfer 10 ml of the solution of Transfer 10 ml of the solution of Transfer 1 ml of the stock solution Transfer 1 ml of the stock solution to
stock to the filter membrane stock to the filter membrane to at least one empty and sterilized at least one empty and sterilized Petri
(pore size 0.45 µm) (pore size 0.45 µm) Petri dish. dish. Add 15-20 ml of Sabouraud
Add 15-20 ml of Trypticasein Soy Dextrose Agar (CAT. 1024) at
< 45ºC Incubation at 20-25 ºC //
Wash each filter membrane 3x100 ml of Buffered Peptone Agar (TSA) (CAT. 1068) at
5-7 days
Water (CAT. 1401) at pH 7.0 < 45 ºC Incubation at 30-35 ºC
// 5 days
MICROORGANISMS MOLDS AND
AEROBIC TOTALS YEASTS TOTALS Select the plates corresponding to the given dilution and
presenting the highest number of colonies lower than 250 for
Transfer the membrane to a Transfer the membrane to TAMC and 50 for TYMC.
Trypticasein Soy Agar (TSA) Sabouraud Dextrose Agar Plates
(CAT. 1068) (CAT. 1024) Find the arithmetic mean of the counts by culture medium.
Incubation at 30-35 ºC // 5 days Incubation at 20-25 ºC // Calculate the number of CFU per gram or milliliter of product
5-7 days

Calculate the number of CFU per gram or milliliter of product

7
B - PLATE COUNT METHODS C - MOST LIKELY NUMBER METHOD (TAMPC)
B.2 SURFACE METHOD

SAMPLE PREPARATION SAMPLE PREPARATION

At a concentration of 1:10, prepare the sample in At a concentration of 1:10, prepare the sample in
Buffered Peptone Water (CAT. 1401) at pH 7.0 Buffered Peptone Water (CAT. 1401) at pH 7.0 and
and homogenize. homogenize.

This stock solution can be supplemented with neutralizersand This stock solution can be supplemented with neutralizers and
surfactants surfactants

MICROORGANISMS TOTAL MOLDS Make two serial dilutions from the stock solution of 1 ml /
AEROBIC TOTALS AND YEASTS dilution in two tubes of 9 ml each of Trypticasein Soy Broth (TSB)
(CAT. 1224)
Transfer at least 0.1 ml of the stock Transfer at least 0.1 ml
solution to a plate of of the stock solution to a Using the initial three dilutions, carry out three further dilutions
Trypticasein Soy Agar (TSA) (CAT. Sabouraud Dextrose Agar plate with at least 3 tubes/dilution in a concentration of 1:10 in
1068) Incubation at 30-35 ºC // (CAT.1024) Trypticasein Soy Broth (TSB) (CAT. 1224) Incubation at 30-35
120 hours Incubation at 20-25 ºC // ºC/120 hours.
5-7 days
Using the initial three dilutions, carry out three further dilutions
with at least 3 tubes / dilution in a concentration of 1:10 in
Trypticasein Soy Broth (TSB) (CAT. 1224) Incubation at 30-35
Calculate the number of CFU per gram or milliliter of product
ºC/ max. 3 days.

CONFIRMATION CONFIRMATION
Tubes with turbidity Tubes without
turbidity
Perform a subculture in the
same broth or on a plate of Determine the Most Probable
Trypticasein Soy Agar (TSA) Number of microorganisms per
(CAT.1068). gram or milliliter of product
Determine the Most Probable
Number of microorganisms by
gram or milliliter of product

8
Escherichia coli

Introduction
This microorganism belongs to the Enterobacteriaceae
family; it is a gram-negative, mobile, non-sporulating
bacillus. In addition, it is lactose-positive and negative
oxidase. Escherichia coli can be distinguished from other
coliforms by its ability to produce indole from tryptophan
or by the production of the ß-glucuronidase enzyme.
These characteristics are used for selective isolation and
confirmation in various analysis processes.

These ubiquitous bacteria are located in the intestine of

humans and warm-blooded animals. Their high presence
in the intestinal tract and feces make it a marker that
indicates bad hygienic practices or fecal contamination Method
during the manufacture or handling of drugs. Their
presence is used as a marker that other pathogenic
organisms of fecal origin may be present, especially in SAMPLE PREPARATION
products meant for oral consumption and natural raw At a concentration of 1:10, prepare the sample using
materials. at least 1 g/ 1 ml of the product to be examined in
Buffered Peptone Water (CAT. 1401) and homogenize.
Today, the tests also contemplate the concentration
of Escherichia coli according to the European This stock solution can be supplemented with neutralizers and
surfactants.
Pharmacopoeia.

PRIMARY ENRICHMENT

Spread 10 ml of the stock solution or the quantity

corresponding to 1 g/1 ml of the product in 100
ml of Trypticasein Soy Broth (TSB) (CAT. 1224) and
homogenize.Incubation at 30-35 ºC // 18-24 hours

SELECTIVE ENRICHMENT
Spread again 1 ml of Trypticasein Soy Broth in 100 ml
of MacConkey Broth (CAT.1210)
Incubation at 42-44 ºC // 24-48 hours

SELECTIVE ISOLATION
Perform subcultures on Agar MacConkey
(CAT. 1052). Incubation at 30-35 ºC // 18-72 hours

Bibliography
RESULTS INTERPRETATION
MacConkey J. H. 5:33. 1905. Joseph Md. State. Dept. Health. The growth of colonies in the medium indicates
Procedures, 1960. European Pharmocopoeia 9.3. the possible presence of E. coli. Confirmation by
Chils, E., and L. A. Allen. 1953. Improved methods for determining identification test. The product passes the test if no
the most probable number of Bacterium coli and of Enterococcus colonies are observed or if identification tests return
faecalis. J. Hyg.Camb. 51:468-477. negative

9
Bile-tolerant
gram-negative
bacteria Method
SAMPLE PREPARATION
At a concentration of 1:10, prepare the sample using at
Introduction least 1 g of the product to be examined in Trypticasein
Soy Broth (TSB) (CAT.1224) and homogenize.
Incubation at 20-25 ºC // 2-5 hours
Infectious diseases have had a decisive influence on
the evolution of human history. They are currently This stock solution can be supplemented with neutralizers and
one of the main mortality causes in the world, being surfactants.
responsible for a significant number of infections. A - TEST FOR THE ABSENCE OF ENTEROBACTERIACEAE
Gram-negative bacteria are characterized by AND OTHER GRAM-NEGATIVE BACTERIA
a double cell membrane, one external and one
cytoplasmic, where a thin wall of peptidoglycan can PRIMARY ENRICHMENT
be found that prevents them from retaining the dye
during Gram stain. The external membrane structure Unless otherwise indicated, spread the volume
comprises an external part consisting of a complex corresponding to 1 g of product in 100 ml of Mossel EE
of lipopolysaccharides whose lipidic part acts as Broth (CAT. 1202). Incubation at 30-35 ºC // 24-48 hours
an endotoxin and is responsible for the pathogenic
capacity of the microorganism. This external
membrane protects them from antibiotics such as SELECTIVE ISOLATION
penicillin, colorants or detergents, a characteristic that Subcultivate in Violet Red Bile Agar With Glucose (VRBG)
makes it possible for them to colonize environments (CAT. 1092) plates Incubation at 30-35 ºC // 18-24 hours
that other microorganisms find difficult to colonize.
Gram-negative bacteria resistant to bile salts include RESULTS INTERPRETATION
Escherichia, Pseudomonas and Aeromonas.
The product passes the test if no colony growth is observed

B - QUANTITATIVE TEST OF ENTEROBACTERIACEAE AND

OTHER GRAM-NEGATIVE BACTERIA

PRIMARY ENRICHMENT

Prepare three tubes with dilutions containing 0.1 g,

0.01 g and 0.001 g of the product to be examined in
10 ml of Mossel EE Broth (CAT. 1202). Incubation at
30-35 ºC // 24-48 hours

Bibliography SELECTIVE ISOLATION

Perform subcultures in Violet Red Bile Agar With
ISO 21528. Microbiology of food and animal feeding stuffs Glucose (VRBG) (CAT. 1092). Incubation at 30-35 ºC //
-- Horizontal methods for the detection and enumeration of 18-24 hours
Enterobacteriaceae.
ISO 8523 Microbiology -- General guidance for the detection RESULTS INTERPRETATION
of Enterobacteriaceae with pre-enrichment. European
Pharmacopoeia 9.0. The growth of colonies indicates a positive result. Write
down the smallest amount of the product that gives
Mossel D.A.A., Visser M. and Cornelissen A.M.R.J App, Bact.
24:444. 1963. a positive result and the largest amount that gives a
negative result. Determine from Table 2.6.13.2 of
European Pharmacopoeia 9.3. European Pharmacopeia

10
Salmonella

Introduction
Salmonella is a very diverse family of microorganisms,
including some 2,300 distinct serotypes. It is one
of the four main causes of diarrheal diseases. This
group of bacteria occurs in the form of gram-negative
bacillus (as they belong to the enormous group of
Enterobacteriaceae), they are facultative anaerobic
and mobile by means of flagella. In addition, they are
glucose fermenters but not lactose fermenters and do
not produce urease.
It is estimated that salmonellosis affects tens of millions
of people worldwide every year and causes more than
100,000 deaths. This type of bacteria is the reason
for millions of hospitalizations worldwide, and this
number may increase up to 30 times if infections that
are not diagnosed are included, as they can usually
cause a normal diarrhea process. It behaves as a
facultative intracellular pathogen that, depending on
the serotype, inoculum, virulence factors expressed by
the strain, the host involved, and the immune status
Method
of the patient can cause from a medium to severe
gastrointestinal infection to a systemic infection. SAMPLE PREPARATION
It is widely distributed in nature and as normal flora of At a concentration of 1:10, prepare the sample using
the intestinal tract in animals and humans. Unlike other at least 10 g / 10 ml of the product to be examined
microorganisms that cause gastrointestinal diseases, in Trypticasein Soy Broth (TSB) (CAT. 1224) and
it may be commonly found in natural raw materials, homogenize. Incubation at 30-35 ºC // 18-24 hours
making it vitally important to research them, especially
those of animal origin. Trypticasein Soy Broth can be supplemented with
neutralizers and surfactants.

SELECTIVE ENRICHMENT
Spread 0.1 ml of Trypticasein Soy Broth
in 10 ml of Rappaport Vassidialis Broth (CAT. 1414)
Incubation at 30-35 ºC // 18-24 hours

SELECTIVE ISOLATION
Perform subcultures on XLD Agar (Xylose Lysine
Desoxycholate Agar) (CAT. 1080)
Incubation at 30-35 ºC // 18-48 hours
Bibliography
RESULTS INTERPRETATION
Rollender, W. U. Beckford; R.D. Belsky, B. Krostoff (1969) All red colonies with/without black nucleus are
Comparison of Xylose Lysine desoxycholate agar and susceptible of Salmonella Confirmation by means of
MacConkey agar for the isolation of Salmonella and Shigella an identification test. The product passes the test if no
from clinical specimens (tech. Bull. Reg. Med. Tech, 39 (1) 8-p).
colonies of the types described are observed or if the
European Pharmacopoeia. 9.3. confirmation tests are negative

11
Staphylococcus
aureus
Introduction
It's a bacterium that belongs to the
Staphylococcaceae family. They are facultative
anaerobic cocci grouped in clusters, gram-positive,
immobile and not sporulating. Among its most
distinctive characteristics is the production of
virulence factors such as coagulases and catalases
that are used for confirmatory tests after isolation.
It is a microorganism widely distributed worldwide
and acts as a commensal in the human epithelium
and mucous membranes. It is often found in
the mouth, blood, mammary glands, intestine,
genitourinary tract, and airways.
It is also an opportunistic pathogen, causing acute
and pyogenic infections which, if untreated, can
spread to the surrounding tissue or to other organs
via bacteremia. Among the types of infections Method
caused by this microorganism are pneumonia,
osteomyelitis, and acute endocarditis, among others.
The presence of Staphylococcus and, particularly, SAMPLE PREPARATION
S. aureus genus in raw material or pharmaceutical At a concentration of 1:10, prepare the sample using
or cosmetic product, indicates that the source of at least 1 g/ 1 ml of the product to be examined in
contamination may be human, i.e. operators. These Trypticasein Soy Broth (TSB) (CAT.1224) / Buffered
microorganisms can be transported through dust, Peptone Water (CAT.1401) at pH 7.2 and homogenize.
skin, clothing, and moisture droplets generated by
moving, talking, and sneezing. This stock solution can be supplemented with neutralizers and
surfactants.

PRIMARY ENRICHMENT

Spread 10 ml of the stock solution or the quantity

corresponding to 1 g/1 ml of the product in
100 ml of Trypticasein Soy Broth (TSB) (CAT. 1224) and
homogenise.
Incubation at 30-35 ºC // 18-24 hours

SELECTIVE ISOLATION
Perform subcultures in Mannitol Salt Agar (MSA)
(Chapman Medium) (CAT. 1062). Incubation at 30-35 ºC
// 18-72 hours

RESULTS INTERPRETATION
Bibliography
The growth of white/white colonies surrounded by
a yellow zone indicates the possible presence of
McColloch Am. J. Vet. Research, 8:173. 1947. Velilla, Faber, Staphylococcus aureus. Confirmation by means of
and Pelczar Am. J. Vet. Research, 8:275. 1947. Chapman, an identification test. The product passes the test if no
G.H. 1945 J. Bact. 50:201-203.
colonies of the types described are observed or if the
European Pharmacopoeia. 9.3. confirmation tests are negative.

12
Pseudomonas
aeruginosa
Introduction
It's a bacterium that belongs to the Pseudomonadaceae
family. They are straight or curved bacilli, strictly
aerobic, gram-negative, mobile and non-sporulating.
In addition, they are positive catalase, using this
characteristic for later identification. This group of
bacteria is capable of producing different pigments such
as pyocyanin and uorescein. Therefore, different culture
media have been developed to promote the production
of these pigments and thus be able to identify the
different species.
This microorganism combines its adaptability to different
ecosystems with a wide variety of virulence factors,
and the spectrum of diseases it can cause ranges from
mild infection to sepsis. During its mechanism of action
it attaches to epithelial cells by means of polar pili
and, once attached, it produces proteases, hemolysins,
exotoxins and endotoxins that damage tissues. Method
The P. aeruginosa infection is often related to water and
aqueous solutions contamination. One of the most severe SAMPLE PREPARATION
infections caused by this microorganism is endocarditis
due to intravenous administration of medications, when At a concentration of 1:10, prepare the sample using
injected into the patient and dissolved or suspended at least 1 g of the product to be examined in Buffered
Peptone Water (CAT. 1401) at pH 7 and homogenize
in watery vehicles. Hence, the importance of detecting
its presence in pharmaceutical products that are to
be administered by inhalation and ocular route or in This stock solution can be supplemented with neutralizers and
aqueous vehicles. Cosmetics may also be susceptible to surfactants.
this type of contamination, especially liquids and creams.
In addition, it can colonize water purification systems
PRIMARY ENRICHMENT
by the formation of biofilms, structures very difficult to
eliminate with the use of salinizing agents. Its remarkable Spread 10 ml of the stock solution or the quantity
resistance to antimicrobials is one of the world's greatest corresponding to 1 g/1 ml of the product in
concerns. 100 ml of Trypticasein Soy Broth (TSB) (CAT. 1224) and
homogenise. Incubation at 30-35 ºC // 18-24 hours

SELECTIVE ISOLATION
Perform subcultures in Cetrimide Agar (CAT. 1102)
Incubation at 30-35 ºC for 18-72 hours

Bibliography RESULTS INTERPRETATION

The growth of colonies in the medium indicates the
King, Ward and Raney. J. Lab. and Clin. Med. 44:301. 1954. Brown possible presence of Pseudomonas aeruginosa.
and Lowbury. J. Clin. Path. 18:752. 1965. Lowbury. J. Clin. Path. Confirmation by means of an identification test. The
4:66. 1951. Lowbury and Collins. J. Clin. Path. 8:47. 1955.
product passes the test if no colonies are observed or if
European Pharmacopoeia. 9.3. the confirmation tests are negative

13
Clostridia

Introduction
They are a class of bacteria belonging to the Firmicutes
phylum class. They are characterized by being mostly
gram-positive anaerobes bacilli. Most genuses do not
form spores, but some do, such as Clostridium, one of
the best known.
These bacteria are widely distributed in nature, mainly
in the soil and in the intestinal tract of many animal
species, including man, where they may also be found
as normal flora on the skin and mucous membranes
of the nasopharynx, pharynx, mouth, urethra, and
vagina. They can cause both endogenous and
exogenous infections, affecting any region of the body,
provided the conditions in the tissues are favorable.
They are fundamentally involved in abscesses of any
organ, colitis, appendicitis, bacteremia, chronic otitis Method
media, crackling cellulitis, dental and oral infections,
endocarditis and endometritis.
SAMPLE PREPARATION
The presence of anaerobic microorganisms, especially
those belonging to the Clostridium genus is usually At a concentration of 1:10, prepare the sample using
found in natural raw materials, powder, starch, etc. at least 2 g/2 ml of the product to be examined in
Trypticasein Soy Broth (TSB) (CAT. 1224) / Buffered
And also in medicinal herbs and phytotherapeutic
Peptone Water (CAT. 1401) at pH 7.2 and homogenize
medications.
This stock solution can be supplemented with
neutralizers and surfactants.

INACTIVATION SPORES
Divide the sample into 2 tubes of at least 10 ml. Heat 1
portion to 80 ºC/10 min and cool quickly. Do not heat
the other portion

PRIMARY ENRICHMENT
Spread 10 ml, separately, of each portion of the stock
solution in 100 ml of Reinforced Clostridial Medium
(CAT. 1007). Incubation at 30-35 ºC // 48 hours

SELECTIVE ISOLATION
Perform subcultures in Columbia Agar (CAT. 1104)
Incubation under anaerobic conditions at 30-35 ºC for
48-72 hours

Bibliography
RESULTS INTERPRETATION
Ellner, Stossel, Drakeford and Vasi. AM J. Clin. Path.
45:502-504. 1966. The growth of colonies (with/without spores) gives a
negative reaction to catalase indicating the presence of
European Pharmacopoeia. 9.3. Clostridia. Confirmation by identification test

14
Candida albicans

Introduction
These are yeasts belonging to the Saccharomycetaceae
family and the Candida genus. These fungi develop in
a unicellular form and are human saprophytic. It is very
common to find them in oral cavities, gastrointestinal
tract and vaginal area. Although they have a very
important function in the fermentation of certain sugars
for the body, there are times that this microorganism
acts as an opportunistic pathogen, being able to cause
superficial or systemic candidiasis in weakened people,
in newborns and in old people with a deficient immune
system.
The products with the highest susceptibility to fungal
contamination are ophthalmic solutions, ointments,
suppositories, salves and in cosmetics, soaps, powders,
and others that contain nutrients rich in carbohydrates
and fatty acids.
It is therefore necessary to analyze these Method
pharmacological products in order to identify the
presence of this microorganism and thus avoid the
consequences of a possible infection by C. albicans in SAMPLE PREPARATION
the consumer.
At a concentration of 1:10, prepare the sample in
Trypticasein Soy Broth (TSB) (CAT.1224) / Buffered
Peptone Water (CAT. 1401) at pH 7.2 and homogenise

This stock solution can be supplemented with neutralizers

and surfactants

PRIMARY ENRICHMENT
Spread 10 ml of the stock solution or the quantity
corresponding to 1 g/1 ml of the product in 100 ml of
Sabouraud Dextrose Broth (CAT. 1205) and homogenize.
Incubation at 30-35 ºC // 3-5 days

SELECTIVE ISOLATION
Perform subcultures in Sabouraud Dextrose Agar plates
(CAT.1024). Incubation at 30-35 ºC // 24-48 hours

Bibliography
RESULTS INTERPRETATION
Beuchat, L.R., J.E Corry, A.D King, Jr. And J.I Pitt (ed) 1986) The growth of white colonies in the medium indicates the
Methods for the mycological examination of food. Plenum Pres, possible presence of Candida albicans. Confirmation by
New York European Pharmacopoeia. 9.3. means of an identification test. The product passes the
Sabouraud, Ann. Dermat and Syphilol 1892-3. Gerog J. Lab.CLin. test if these colonies are not observed or if confirmatory
Med. 67;355 1953. tests return negative

15
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