Criteria for Classification of Bacteria
1.
2.
3.
4.
5.
Growth on Media
Bacterial Microscopy
Biochemical Tests
Immunologic Tests
Genetic Features
1. Growth on Bacteriologic Media
In contrast to viruses and most parasites, many bacterial pathogens can
be isolated on solid media rich in metabolic nutrients and carbon source.
Bacteriologic Media may be Enrichment media, Selective media,
Differentiative Media.
Upon culture, some bacteria produce characteristic pigments, and others
can be differentiated on the basis of their complement of extracellular
enzymes; the activity of these enzymes often can be detected as zones of
clearing surrounding colonies., eg, zones of haemolysis in Blood agar
medium containing red blood cells.Figure 1. Blood Agar Plates contain
mammalian blood (usually sheep or horse or human), typically at a
concentration of 5–10%. Blood Agar Plates are enriched, differential
media used to isolate fastidious organisms and detect haemolytic activity.
 alpha haemolysis (α-haemolysis) : partial haemolysis, agar under the
colony is dark and greenish.
 Beta haemolysis (β-haemolysis): complete haemolysis of red blood
cells, the media around and under the colonies appears lightened, clear
and transparent.
 Gamma haemolysis(γ haemolysis): non haemolysis, the agar under and
around the colony is unchanged.
1
Many of the members of the Enterobacteriaceae can be differentiated on
the basis of their ability to metabolize lactose such as lactoce fermentant
or lactose non fermentant on MacConkey agar , Figure 2.
MacConkey agar is designed to selectively isolation of Gram-negative
bacteria and differentiate them based on lactose fermentation into lactose
fermentate(pink colony), e.g, E coli, and lactose non fermentate(pale
colony), e.g, Salmonella sp.
MacConkey agar contains crystal violet and bile salts as inhibitors to the
growth of gram-positive bacteria which allows for the selection and
isolation of gram-negative bacteria.
2
Mannitol Salt Agar (M.S.A.)
Mannitol salt agar or M.S.A. is important in medical laboratories by
distinguishing pathogenic microbes in a short period of time. M.S.A is a
commonly used as a selective and differential Medium in microbiology.
This medium encourages the growth of a group of certain bacteria while
inhibiting the growth of others because it contains a high concentration
(about 7.5%-10%) of salt (NaCl), which making it selective for Grampositive bacteria, Staphylococcus, since this level of salt is inhibitory to
most other bacteria. It is also a differential medium for mannitol
fermenting staphylococci, containing carbohydrate mannitol and the
indicator phenol red a pH indicator for detecting acid produced by
mannitol-fermenting staphylococci. S. aureus produces yellow colonies
with yellow zones, whereas other coagulase-negative staphylococci
3
produce small pink or red colonies with no colour change to the medium.
If an organism can ferment mannitol, an acidic product is formed that
causes the phenol red in the agar to turn yellow. It is used for the
selective isolation of presumptive pathogenic Staphylococcus species.
Expected results
 Gram (+) Staphylococcus: fermenting mannitol: medium turns yellow
(e.g. S. aureus)
 Gram(+) Staphylococcus: not fermenting mannitol, medium does not
change color (e.g. S. epidermidis)
 Gram(+) Streptococcus: inhibited growth.
 Gram( -): inhibited growth.
4
2. Bacterial Microscopy
Historically, the Gram stain, together with visualization by light
microscopy, has been among the most informative methods for classifying
the bacteria. Depend on the differences in the structure of their cell walls,
Gram stain divides bacteria into gram negative and gram positive
bacteria. In addition, Bacteria are classified by direct examination with the
light microscope through it's morphology and aggregation. Under
microscpe, Staphylococcus aureus has gram-positive spherical
cells(cocci) aggregate in grape-like clusters.( figure 3).
5
3. Biochemical Tests
A diverse range of biochemical tests are known for the identification of
certain metabolisms and to differentiate among bacteria. In most cases,
detection is based on the reaction of an enzyme with a certain substrate
and the results of these tests are seen by color change.
Biochemical Tests for Staphylococcus aureus
1) Catalase test:
Catalase test is a common enzyme found in staphylococci. Catalase
catalyzes the decomposition of hydrogen peroxide (H2O2) to water and
oxygen. It is a very important enzyme in protecting the cell from
oxidative damage by reactive oxygen species. One catalase molecule can
convert millions of hydrogen peroxide molecules to water and oxygen
each second. Catalase test is used to differentiate Staphylococci from
Streptococci. Staphylococci and Micrococci are catalase-positive(figure
4), while Streptococcus and Enterococcus are catalase-negative.
6
7
2) Coagulase test
coagulase test has traditionally been used to differentiate Staphylococcus aureus
from coagulase-negative staphylococci. Coagulase is an enzyme produced by the
bacteria that enables the conversion of fibrinogen to fibrin. In the laboratory, it is
used to distinguish between different types of Staphylococci isolates. Importantly,
S. aureus is generally coagulase-positive. S.aureus produces two forms of
coagulase: bound coagulase and free coagulase. Bound coagulase, otherwise
known as “clumping factor” can be detected by carrying out a slide coagulase
test, and free coagulase can be detected using a tube coagulase test( figure 5).
Procedure of Coagulase test
1. Slide Coagulase Test






A slide coagulase test is run with a negative control,(why? To rule out
autoagglutination).
Two drops of saline are put onto the slide labeled with sample number, Test
(T) and control (C).
The two saline drops are emulsified with the test bacteria using a wooden
stick.
A drop of plasma, is placed on the inocu-lated saline drop corresponding to
test, and mixed well, then the slide is rocked gently for about 10 seconds.
((usually, rabbit plasma anti-coagulated with EDTA, why? To prevent the
plasma auto-coagulation before the test))
Positive result: clumping would be observed in the plasma within 10
seconds, with no clumping in the saline drop.
Negative result: no clumping will be observed. SO, a tube test should
follow as a confirmation..
The Clumping in both drops is an indication of autoagglutination, so a tube
test should be carried out.
8
Procedure of Coagulase test
2. Tube Coagulase test
Staphylococcal pigments
Some strains of S. aureus are capable of producing Staphyloxanthin.
Staphyloxanthin is a golden-coloured carotenoid pigment(figure 6). This
pigment acts as a virulence factor, primarily by being a bacterial
antioxidant which helps to evade the reactive oxygen species which the
host immune system uses to kill pathogens.
Mutant strains of S. aureus modified to lack staphyloxanthin are less
likely to survive incubation with an oxidizing chemical, such as
hydrogen peroxide, than pigmented strains. Mutant colonies are quickly
killed when exposed to human neutrophils, while many of the pigmented
colonies survive. In mice, the pigmented strains cause lingering
9
abscesses when inoculated into wounds, whereas wounds infected with
the unpigmented strains quickly heal. These tests suggest the
Staphylococcus strains use staphyloxanthin as a defence against the
normal human immune system. Drugs designed to inhibit the production
of staphyloxanthin may weaken the bacterium and renew it's
susceptibility to antibiotics.
37°
10
C 37°C
11