University of Juba
School of Medical Laboratory Sciences
MMLM 422 Systematic Bacteriology II 4th Year
Conventional Methods for Bacterial Identification.
Phenotypic Characteristics
Date:25.11.2025 Michael Lasuba
Phenotypic Characteristics
Difference between genotype and phenotype
• The fundamental difference between genotype and phenotype is
that:
• The genotype is an organism's inherited genetic makeup (DNA
sequence),
• While the phenotype is its collection of observable
characteristics resulting from the interaction of its genotype with
the environment.
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Methods used for the identification of microorganisms
• The primary methods used for the identification of microorganisms,
categorizing them into two main approaches: phenotypic characteristics and
genotyping/molecular identification.
• Phenotypic Characteristics: This traditional approach involves observing
physical and biochemical traits.
• Genotyping and Molecular Identification: Modern techniques analyze the
organism's genetic material (DNA/RNA).
• Specific Phenotypic Methods: These include appearance (colonial and
microscopic morphology), biological activity (enzyme production, sugar
fermentation), and immunophenotyping (serotyping).
• Application: These methods are crucial in fields like diagnostic medical
microbiology and food safety for accurate identification of pathogens 5
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1. Oxygen requirements experiment
• 1. Oxygen requirements experiment
• Answer: (a) Obligate aerobe
• The image likely depicts a fluid thioglycollate medium test, which is used to
determine the oxygen requirements of different microorganisms.
• In the tube on the left, growth (indicated by the red dots) is concentrated at
the top surface. This indicates that the organisms require oxygen to
grow. This type of organism is an obligate aerobe.
• In the tube on the right, growth is concentrated at the bottom of the tube,
where oxygen is absent. This indicates that the organisms cannot grow in the
presence of oxygen. This type of organism is an obligate anaerobe.
• The text snippets mention "cells for O2" and "super oxide dismutase," which
are related to oxygen metabolism and defense against reactive oxygen
species, further supporting this interpretation.
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2. BIOLOGICAL ECTIVITIES
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2 H202 -- catalase--
-- 2 H20 + O2
bubbles
Gelatinase / Proreinase Principal
Gelatinase is an exoenzyme that hydrolyzes gelatin (a protein derived from
collagen) into smaller polypeptides and amino acids, which are then transported
into the cell for metabolic use. The production of this enzyme helps bacteria obtain
nutrients from their environment.
The gelatin hydrolysis test is used to detect the presence of this enzyme:
Positive Result: The gelatin medium in the test tube liquefies, even after
refrigeration.
Negative Result: The medium remains solid after refrigera
Gram-Positive Bacteria
Positive for gelatinase:
Bacillus subtilis
Clostridium species
Enterococcus faecalis, where the enzyme can contribute to virulence
Negative for gelatinase:
Staphylococcus aureus strains generally do not liquefy gelatin (though some strains
might)
Micrococcus roseus
Gram-Negative Bacteria
Positive for gelatinase:
Pseudomonas aeruginosa
Serratia marcescens
Flavobacterium species
Negative for gelatinase:
Many members of the Enterobacteriaceae family, such as Escherichia coli, are often
gelatinase-negative, although the test is a common differentiating factor among family
members.
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Principle of the Litmus Milk Test
• The medium contains lactose (milk sugar), casein (milk protein), vitamins, minerals, and the
litmus indicator. Bacteria can metabolize these components in various ways:
• Lactose Fermentation: Bacteria ferment lactose to produce organic acids (like lactic acid),
which lowers the pH. The litmus indicator turns pink or red in an acidic environment.
• Casein Metabolism (Peptonization/Digestion): Some bacteria break down the casein protein
(proteolysis), releasing ammonia or creating a clear, grayish, watery fluid. This raises the pH,
turning the litmus indicator blue or purplish-blue.
• Reduction (Decolorization): The litmus indicator itself can be reduced (decolorized) by some
bacteria in an anaerobic environment, causing the medium to turn white.
• Curd Formation (Clotting): Acid production or rennet enzyme production can cause the milk
proteins to coagulate, forming a solid curd (clot).
• Acid curd: A hard, insoluble curd with a clear supernatant (whey).
• Rennet curd: A soft, often alkaline curd that may undergo peptonization later.
• Gas Production: Some bacteria produce gas during fermentation, which may be visible as
bubbles or cracks in a solid curd (referred to as "stormy fermentation").
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Examples of bacteria
• Examples of bacteria demonstrating these reactions include Lactobacillus
acidophilus (acid and acid clot),
• Pseudomonas aeruginosa (peptonization), and Clostridium perfringens (gas
production).
• Gram-positive bacteria like Lactobacillus and Streptococcus are known for
acid and clot formation, while some Gram-negative Enterobacteriaceae can
reduce litmus.
• The litmus milk test is a differential but not a definitive test. Results may
require confirmation with other methods like Gram staining.
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Triple Sugar Iron Media (TSI)
• The medium contains three sugars (glucose, lactose, and sucrose) and a pH
indicator (phenol red), which turns yellow in the presence of acid produced by
fermentation.
• The slanted shape provides both aerobic (slant) and anaerobic (butt)
environments, allowing for the detection of different metabolic activities.
• A red slant and yellow butt (e.g., tubes 1, 2, 3) indicate that only glucose was
fermented.
• A yellow slant and yellow butt (e.g., tubes 4, 4A) indicate fermentation of all
three sugars.
• Blackening of the medium (e.g., tubes 3, 5) indicates the production of
hydrogen sulfide gas.
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Analytical Profile Index (API) System
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Analytical Profile Index (API) System
• Key information about the product:
• Manufacturer: bioMérieux.
• Function: Rapid and precise identification of Gram-negative bacteria based
on their metabolic activity and biochemical reactions.
• Contents: Each strip contains 20 small reaction tubes with dehydrated
substrates.
• Usage: A bacterial sample is inoculated into the tubes, incubated, and color
changes are read and converted into a 7-digit code for species identification
via a database.
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Minimum inhibitory concentration (MIC)
• The broth dilution method, a technique in microbiology used to determine the
minimum inhibitory concentration (MIC) of an antimicrobial agent against a
specific microorganism. The MIC is the lowest concentration of the agent that
prevents visible growth of the organism.
• Essential information regarding the process:
• Serial dilutions of an antibiotic are prepared in a liquid growth medium
(broth).
• Standardized bacterial cultures are added to the tubes.
• After incubation, the tubes are examined for visible bacterial growth
(turbidity).
• The MIC is identified as the lowest antibiotic concentration where no visible
growth is observed.
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Microdilution microwell plate
• The image shows a microplate, also known as a microtiter or microwell plate,
which is a standard laboratory tool with multiple small wells used as test
tubes.
• These plates typically have 96 wells and are made from transparent
polystyrene.
• They are often tissue culture treated, with flat bottoms that promote cell
growth for adherent cell cultures.
• The plates are commonly sterilized by gamma radiation and individually
wrapped to ensure sterility.
• Inscribed alphanumeric codes on the sides and between the wells help in
identifying and tracking samples.
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E-test (epsilometer test)
• The image is a slide titled "E-Test for fastidious microorganism" and shows an
E-test strip on a blood agar plate. The E-test (epsilometer test) is a method
used in microbiology to determine the minimum inhibitory concentration (MIC)
of an antibiotic needed to inhibit the growth of a microorganism, particularly
fastidious ones. Blood agar is an enriched medium containing blood that
provides essential nutrients for fastidious bacteria.
• Essential information:
• The slide title is "E-Test for fastidious microorganism".
• The image displays an E-test strip placed on a blood agar plate.
• The E-test is a technique for determining the Minimum Inhibitory
Concentration (MIC) of an antibiotic.
• Blood agar is a nutrient-rich medium used to grow bacteria that have specific
nutritional requirements
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