Microbiology Basics - Cells, Staining and Identification

MICROBIOLOGY - continue to undergo revolutionary changes through the impact of
new development in cell, molecular and environmental biology. This compilation of
knowledge aims to provide an understanding of the nature and diversity of
microorganism and of their place in the world.
Exciting fields in the study of Microbiology such as: plasmid genetics, macromolecular
synthesis, membrane functions and biogeochemistry are not however covered here.
Here, we are dealing more on the very basics facts about these microorganisms. That
these microorganisms happened to be a large and diverse group of free-living forms
that exist as single cells or cells clusters. That these microbial cells are distinct from the
cells of animals and plants which are unable to live alone in nature but can exist only as
part of multicellular organisms. That a single microbial cell can generally carry out its
life processes of growth, respiration and reproduction independently of other cells.
The cells Is functional unit of all living matter. A single cell is isolated from other cells by
cell wall or membrane and containing within it a variety of materials and subcellular
structures. All cells contain proteins, nucleic acids, lipids and polysaccharides.
These microorganisms are so diverse that it is useful to give them names. They can be
defined by a set of characteristics which make them unique from the other organisms.
Microbiologists use the binomial system of nomenclatures. Genus and species name
are always used together to describe a specific type of organisms. Usually the name
come from Latin and Greek.
The existence of creatures too small to be seen by the naked eyes had long been
suspected and the first person to see microorganisms in any detail was the Dutch
microscope builder, Antoni van Leeuwenhoek, who used for the occurrence of
contagious disease, although it was not until the work of Robert Koch that the gem

theory of disease was placed ona firm footing. He carried out several experiments and
formulated the following criteria is order to be able to prove that a specific type of
bacterium or microbe cause a specific disease. These are now called the Koch’s
Postulates:
1. The organism should always be found in animals suffering from the disease and
should not be present in healthy individuals.
2. The organisms must be cultivated in pure culture away from the body of the
animal.
3. Such a culture, when inoculated into susceptible animals, should initiate the
characteristics disease symptoms.
4. The organisms should be re-isolated from these experimental animals and
cultured again in the laboratory, after which it should still be the same as the
original organism.
These Koch’s postulates provided the tremendous impetus in the development of the
use of laboratory culture in the science of Microbiology.
It is very important to remember however that NOT ALL diseases are caused by
microbes. These are conditions that are inherited, or are caused by deficiencies in the
diet or some harmful influences in the environment. To produce disease, the microbe
must infect a susceptible host and NOT ALL hosts are susceptible. The state of the
health of a host generally influence the outcome of the interactions between the human
host and the infecting microbe.

One may therefore think that because SOME microbes can cause disease, all microbes
are harmful. This is however far from truth because MOST microbes are beneficial to
man or at least, are harmless to man.
MICROBIOLOGY has come a long way from the days of Koch and Pastuer. Today, it is
one of the most sophisticated of the biological sciences and in general has influenced
biology to a great extent. Many special laboratory equipments are now available for the
study of the microorganisms. The knowledge and understanding of molecular biology
actually came from the study of microbes.
MICROBIOLOGY
It is the science that deals with the study of microorganisms and their activities.
It is concerned with their form, structure, reproduction, physiology, metabolism and
identification. The science also include the study of their distribution. The science also
includes the study of their distribution in nature, their relationship to each other as well
as to and their physical and chemical changes they make in their environment.
Generally speaking, the microorganisms are composed of minute unicellular
organisms with both plants and animal characteristics. Those microbes were assigned
by E.H. Haeckel to the Kingdom PROTISTA. The protists are characterized by their
lack of definite cellular arrangement as well as a lack differentiation of cells for specific
metabolic function. They are further divided into:
1. Procearyota or lower protists – organisms which are provided with a primitive
type of nucleus, lacking a clearly defined membrane. The nucleus
is rudimentary with a single chromosome. There is no mitotic
apparatus. Under Procaryota are:
a. Schizophycene which includes the blue green algae.
b. Schizomycetes which includes the bacteria and the pleura-
pneumonia like organism (PPLO)

c. Microtatobiota which includes the rickettsine, chlamydiae and the
viruses.
2. Eucaryota or higher protists – organisms which possessa well-defined nuclear
membrane, chromosomes and they exhibit mitotic cell division. Under the
Eucaryota are algae, protozon, fungi and the slime molds.
Laboratory identification of microorganisms:
1. Morphological study – which can be done whether the organisms are in the living or
fixed state.
a. Living state: a.1. wet mount
b. 2. Hanging drop method (HDM)
b. Fixed state: fixed and stained preparation.
Staining is the process artificially coloring the microorganisms with
dyes. The advantages of this procedure are: (1) cells are made
more clearly visible and (2) differences between cells can be
demonstrated. The essential steps in this procedures were
(1) Preparation of the smear
(2) Fixation and (3) application of staining solution/s.
The different kinds of staining are:
1. Simple staining where the coloration of organisms is done by the application of a
single solution of stain.
2. Differential staining is slightly more elaborate then the above, since the organisms
are exposed to more than one staining reagents. In this technique, differences
between cells and/or parts of the cells could be elicited. This is exemplified by the
Gram stain, the Acid-fast stain and the Giemsa stain.
3. Special staining are the procedures for the demonstration of the different structures
within or exterior to bacterial cell wall.
This includes anyone of the following:

3.1. Cell wall staining - Dyar method
3.2. Capsular staining - Tyler capsule staining
- Hiss capsule staining
- Anthony’s staining
- Welch’s capsule staining
3.3. Spore staining - Heat and acetic acid method
- Dorner’s spore stain
3.4. Flagellar stain - Gray’s method
- Leifson method
- Caesares Gil method
- Loeffler’s method
- Van Ermenger method
3.5. Metechromatic granules - Loeffler’s Alkaline Methylene Blue
method (LAMB)
- Meisser method
- Albert’s method
4. Indirect/Relief/Negative staining is a technique where the organisms are not stained
but the background:
4-1. India ink method 4.2. Nigrosine method

GRAM staining
This is one of the most important widely use differential staining methods in
Microbiology. In this process, the fixed bacterial smear is exposed to the following
solutions in the order as listed:
1. Crystal violet - the primary dye. It purpose is to stain all the organisms in the
smear.
2. Gram’s iodine - the mordant which enhances or strengthens the union between
the primary dye and its substrate
3. 95% Ethyl alcohol – the decolorizer which dissolves and removes the primary stain
from the cells.
4. Safranin - the counterstain.
Bacteria stained by this method fall into two groups:
(1) gram-positive – those that retain the crystal violet hence appear deep violet in
color
(2) gram-negative – those that lose the crystal violet by decolorization therefore
absorb the safranin when counterstained, hence appear red in
color.
The gram-positiveness or negativeness of a microorganism was found to be due to the
chemical make up of the cell wall. One theory suggests that microorganism are positive
because of a special magnesium-ribonucleic acid-protein-carbohydrate complex that
forms an insoluble substance with the crystal violet and iodine. The gram-negativeness
on the other hand is believed to be due to the high lipid content of the cell wall of the
organisms.
ACID-FAST staining is especially used for the Mycobacteria. The fixed bacterial smear
is subjected to the following reagent in the orders as listed|:
1. carbol-fuchsin - the primary stain.
a. Ziehl-Neelsen method – carnol-fuchsin is steamed for 5-6 minutes.

b. Kinyoun method also known as cold method because carbol-fuchsin is not
steamed but remained flooded from 30 – 60 minutes.
2. Acid-Alcohol – the decolorizer
3. Methylene blue – the counterstain.
Bacteria stained under this method are classified into:
(1) Acid – fast – those that resist- decolorization by the acid alcohol meaning they
retain the carbol-fuchsin hence appear red.
(2) Non-acid-fast are those that are decolorized by the acid alcohol and
counterstained by the methylene blue hence appear bluish.
The acid-fastness of the organisms is believed to be due to the presence of
mycolic acid in the cell wall.
II. Cultural Methods – whereby organisms are culticated under laboratory conditions.
This was made possible by the discovery of the different nutritional
requirements of particular bacteria. The discovery resulted in the
development of a wide variety of culture media.
Culture media – considered to be the artificial soil of the microorganisms
because they contain the nutritional and environmental
requirements for bacterial growth.
Culture/Colony – crop or the growth of organisms bacteria are
grown artificially for the following reasons:
(1) Isolation and identification of microorganism especially from specimens collected
from patients
(2) Quantitative estimation where specimens are cultured in known volumes and the
number of bacterial colonies can be counted.
(3) Antimicrobic testing. The suspectibility of bacteria to antibiotics is tested by growing
the microorganisms in the presence of these various anti-microbial substances.

(4) Sterility testing of operating room items, biological products and blood from blood
banks require sterility checking.
(5) Production of biological products – in the production of vaccines and anti-toxins,
laboratory isolated – organisms under controlled conditions are necessary to obtain
therapeutic levels.
(6) Stock cultures are kept for teaching and control purpose. These are pure culture of
organisms used as a source of supply for industry and research.
Culture may be:
(1) Pure – when it is made up of organisms belonging to one species
(2) Mixed – when made up of different species.
All living organisms have a set of physiological requirements for growth and so with our
microbes. With these various requirements, microorganisms may also be classified
according to their specific needs.
A. Nutritional requirement:
1. Lithotrophs (Autotrophs) - organisms that depend in inorganic matter for
growth
2. Organotrophs (Hetarotrophs) – organisms that depend on organic matter for
growth
B. Oxygen requirement:
1. Aerobes
a. obligate – when they survive mainly in the presence of atmosphere oxygen
b. facultative – are basically anaerobic organisms but are able to survive in the
presence of atmospheric oxygen
2. anaerobes
a. obligate – organisms that survive mainly in the absence of atmospheric
oxygen

b. facultative - are basically aerobic but can survive even in the absence of
atmospheric oxygen
3. microaerophiles are organisms that survive in the presence of a small amount of
atmospheric oxygen.
C. Thermal requirement:
1. Psychrophilic – when organisms grow at a temperature below 10’,C also known
as the cold-loving organisms
2. Mesophilic – when organisms grow around 37’C
3. Thermophilic – when organisms grow at 45’C and above.
Most pathogenic organisms grow as mesophiles.
D. pH requirement:
1. acidophilic – when organisms prefer an acidic environment
2. basophilic – when organisms prefer an alkaline environment
E. Moisture
F. Osmotic pressure
Based on these requirements, organisms therefore must be:
1. Inoculated on a medium of suitable nutritional content
2. Incubated in a appropriate physical conditions
Classification of Culture Medium (pl.media)
A. Based on consistency : 1. liquid
2. semi-solid
3. solid
B. Based on composition:
1. Synthetic – when the components of the media are chemically defined
2. Non-synthetic – when components are not chemically defined
C. Based on how media is dispensed :

1. Tube – slant
butt
butt and slant
2. Plated
D. Based on use:
1. Simple – media that support the growth of many common heterotrophs
e.g. nutrient agar/broth
2. Enrichment media – when additional nutrients are added to support the
growth of fastidious organisms.
e.g. addition of blood, serum or extracts
blood Agar plate (BAP)
3. Differential media - when certain reagents or chemicals are incorporated to
favor growth resulting in the differentiation between types of bacteria.
e.g. Blood agar plate – allowing the observation on the different
hemolytic activities of different organisms
4. Selective media – when specific chemical substances withour inhibiting
others. e.g. Eosin-Methylene Blue (EMB)
It favors the growth of Gram negative rods but inhibit growth of
Gram positive organisms
5. Special/Specific media are those made up of prescribed or special
components usually used in the assay of vitamins, amino acids, antibiotics
and disinfectants.
III. Biochemical study – Microbial cells are capable of performing a multitude of
chemical changes which are exceedingly – complex. These are made possible
through the enzyme system within the cell. Some important and common changes
may be:
1. Carbohydrate fermentation 4. Hydrolysis of urea
2. Catalase reaction 5. Nitrate reduction
3. Oxidase reaction 6. Liquefaction gelatin
IV. Serological Study – detects the presence of antibodies in the patients serum
against specific antigens like:
1. Widal test

2. Weil-Felix test
3. Serological test for syphilis (STS) e.g. Rapid Plasma regain (RPR)
TPHA (Treponema pallidum Hemagglutination)
VDRL (Veereal Disease Research Laboratory)
FTA – Abs (Flourenscent Treponema antibodies – Abdsorption)
Wasserman test – complement fixation test (CFT)
4. Hemagglutination Inhibition (HI) for some viral conditions
e.g. Rubelle titer in German measles
5. Anti – Streptolysin “O” Titer (ASOT)
6. Fluorescent antibody Test (FAT) in gonorrhea and still many others.
V. Animal Inoculation test may also be done in certain infections.

BACTERIA are unicellular microscopic organisms.
Normal habitat - soil, water, air, milk, food, animals
Normal flora - skin, nose, nasopharynx, mouth, intestinal tract genitals,
genitor-urinary tract
Size – unit of bacterial measurement is micron (pl. micra) which is equivalent to
1/1000 mm or 1/25,000 inches. Bacteria commonly studied in the
loaboratory measure approximately 0.5 to 1 micron by 2 to 5 micra. This
measurement however may vary in either direction.
Reproduction – binary or transverse fission, in which a single call divides into two
after developing a transverse cell wall
Shape and arrangement:
1. Spherical or ellipsoidal – cocci (singular, coccus)
Maybe arranged : a. singly
b. in pairs – diplococcic
c. in chain – streptococci
d. in bunch or cluster – straphylococci
e. in group of 4’s
2. Cylindrical or rod-like bacilli (singular, bacillus)
Maybe arranged: a. singly
b. in pairs – diplobacilli
c. in chain – streptcoacilli
3. Spiral – shaped – spirilla (singular, spirillum)
Predominantly individual cells, which may exhibit striking
differences in length, number and amplitude of sprirals as well as
rigidity of cell walls.
Bacterial Structures (Anatomy)
There are certain definite structures inside and outside the cell wall. Some
structures are naturally common to all bacteria while other structures are restricted to
certain species. These bacterial structures includes the following:
1. Capsule is the thickened slimy or gelatinous substance forming covering

layers or envelope around the cell. It serves the bacteria as a protective
covering that can delay the digestion of bacteria by phagocytosis.
The capsule is associated with the virulence of the bacteria.
2. Cell Wall – Beneath the extracellular capsule is a rigid structure which gives
and maintains the shape of the organism. This is made up of complex
polymeric substances known as the peptidoglycan primary muramic acid or
murein combined with amino acids, amino sugars, carbohydrates and lipids.
If the cell walls is removed, what could be left behind is either:
a. Protoplasts – when the c viability of the cells is retained
b. Spheroplasts – when part of the cells is synthesized
3. Cell Membrane or cytoplasmic membrane or protoplasmic membrane
Immediate beneath the cell wall is a thin membrane that is semi-permable,
composed of phospholipids and proteins. It was found to be responsible for:
a. Selective permeability and transport of solute into the cell
b. Electron transport and oxidative phosphorylation
c. Excretion of hydrolytic exo-enzymes
d. Serves as the site of enzymes and carrier molecules which helps in the
biosynthesis of DNA, cell wall polymers and membrane lipids.
4. Nucleus – appears obscure since bacterial cells do not contain nucleus which
characteristic of higher plants and animals. Possess strends of the DNA
molecule.
5. Cytoplasm – found to be rich in DNA and RNA rich particles in combination
with proteins are called ribosomes which contain the bacterial enzymes
responsible for protein biosynthesis.
6. Cytoplasmic inclusions: occurs largely in connection to accumulated
food substances. Appears of these inclusions is also related to the age of the
bacterial cells.

a) Metachromatic granules or Babes – Ernst granules or volutin
granules or Much granules derived from inorfanic phosphate
now largely made of polymetaphosphate.
b) Granulose – polymers of glucose which started as a source of
carbon
c) Sulfur granules – excess hydrogen sulfide can develop into
elements sulfur
7. Flagella – extremely thin hairlike appendages that protrude through the cell
wall. They are mainly composed of protein sub-units. They are responsible
for the motility of the organisms. Not all bacteria are flagellated therefore in
terms of the flagella the bacteria may be:
(1) Motile or (2) Non-motile
Bacterial cells can also be classified according to the flagella as follows:
a. Atrichous – when the bacterial cell has NO flagellum
b. Monotrichous – when the bacterial cell has one flagellum at one polar
ends
c. Amphitrichous – when bacterial cell has one flagellum at the both ends
d. Lophotrichous – when the bacterial cell has a tuft of flagella at one end
e. Peritrichous – when the bacterial cell has flagella all over its body
8. Pili/fimbriae–found in some gram negative bacteria as rigid surface
filamentous appendages. In comparison to that flagella, they are smaller,
shorter and more numerous. They are only seen under the electron
microscope. They do not have function for motility but:
a. They facilities the adherence of the bacteria to tissues from which they
can derive nutrition.
b. They have sexual functions thus are involved in bacterial conjugation.
9. Spores (Endospores) – some bacteria are able to transform into small
ovoidal or spherical bodies that are highly resistant in the presence of

adverse conditions. When the favorable conditions returns, the spore can
germinate and produce a vegetative cell. Spores forming-bacteria belong to
general Bacillu and Clostridia. Location of the spore may be:
a. Central
b. Terminal
c. Sub-terminal
Bacteria maybe:
1. Pathogenic – related to production of disease process in man
2. Non-pathogenic or saprophytic. As saprophytes they are involved in the
following activities:
a. Decomposition of deed matter
b. Soil fertilization
c. Sewage purification
d. Useful industrial processes e.g. formation of coal production of methane,
tobacco industry, leather industry, production of antibiotics, etc…
e. Useful in food manufacture – production of alcoholic beverage, cheese
manufacture, production of buttermilk, yogurt etc..
Pathogenic bacteria
Mode of transmission:
1. food and water-borne e.g. Salmonellae, Vibric
2. soil – borne e.g. Clostridia
3. air – borne – droplet e.g. Mycobacteria, Diphtheria, cold virus
4. Arthropod – borna e.g. Denrue virus, Borrelia, Rickettsiae
5. Sexually-transmitted/veneral e.g. Nisseria gonorrhea
Treponema pallidum
Herpes progenitalis
Hemophilus ducreyi
Chlamydia lymphogranulomatis
Calymmatobacterium inguinale
Condyloma acuminate
6. Animal – borne e.g. Rabies virus, Bacillua anthracis
Streptobacillus moniliformis

7. Fomite – borne/ Nosocomial – from materials infected or contaminated by a sick
person like bedsheets etc..
VIRULENCE – the capacity of a microorganism to produce disease
Factors influencing the virulence :
1. Toxin production : exotoxins and endotoxins
2. Enzymes elaboration
3. Capsule possession
Maintenance of a microbial infection is possible when:
1. there is a disease agent (pathogenic – organisms)
2. there is a suitable reservoir host e.g. sick person
3. there is a susceptible host
4. the organism can exhibit a satisfactory mode of entry
5. the organism can exhibit an accessible portal of exit
6. there is an appropriate means of transmission to a new host
Bacterial Growth Curve
Phases (Periods) of Growth :
1. Lag phase – also known as the phase of rejuvenesence of physiological growth.
This is the period of adjustment of the organism to the new
environment such that there is no or very little multiplication.
2. Logarithmic phase – also known as the exponential phase
- the bacterial cells are most active during this phase
- organisms grow and multiply at its maximum rate
3. stationary phase – also known as the period of equilibrium
- the number of growing cells equal the number of dyong
cells
- the population of cells remains constant at this time
4. decline phase – also known as death when bacterial cells are dying faster than
they are reproduced.
- there is cessation of multiplication
Bacterial Control:
Sterilization –total inactivation of all forms of bacterial life in terms of the ability of
the bacteria to reproduce

Methods of sterilization:
I. Physical Methods
1. Use of Heat
A. Moist Heat B. Dry Heat
1. Autoclave/Steam under 1. flaming with Bunsen burner
pressure
121 0X 15 lbs pressure 2. Oven at 1500 – 1800C
15 – 20 minutes for 30 – 60 minutes
2. Arnold’s sterilization 3. Incineration/Burning
or Tyndallization or fractional 300-4000C
sterilization 4. Cremation – burning
65-1000C 15-20 minutes for of dead bodies
three consecutive days
3. Inspissation – process of thickening
through evaporation at 80-900C for two
hours in three consecutive days.
4. Boiling at 1000C for 20-30 minutes
5. Pasteurization – killing organisms in a product like
milk or fruit juice by heating under controlled
temperatures that do not materially change the
natural palatability of the substance
a) Low temperature holding (LTH) or Vat pasteurization
done at 1450F or 62.80C for 30 minutes
b) High temperature short time (HTST)
done at 1610F or 71.70C for 15 seconds
2. Radiation – through the use of ultra-violet rays (UV)
- preferred for space sterilization like nurseries, operating
room, inoculating hoods etc.

3. Filtration – usually used in the sterilization of media that are destroyed by
heat. Anyone of the following is useful:
a. Berkefeld – made up of infusorial earth
b. Chamberland – made of porcelain
c. Seitz*Works – made up of metal and asbestos
d. Pyrex – made up of sintered glass
e. Membrane/Millipore – made up of ester membrane
II. Use of Chemicals
1. Disinfectants
2. Dyes
3. Detergents
4. Acids, Salts
5. Vapors like formaldehyde
6. Anti-microbials/Antibiotics
GRAM POSITIVE COCCI
STARPHYLOCOCCUS are Gram + occi that often occur in irregular grape-like
clusters. Few strains are capsulated. Some are aerobic and some are facultatively
anaerobic. They grow best 350C. growth is improved in the presence of blood. They
also grow on nutrient agar and on trypticase soy wroth. When grown on agar, usually,
they develop characteristic colors.
Important species :
1. Staphylococcus aureus
2. Staphylococcus epidermidis
3. Staphylococcus saprophyticus
Staphylococcus aureus
As a saprophyte, it is found on the skin, mucous membrane, nose, mouth and
intestines.
As a pathogen:
1. It can cause suppurative conditions like boils, carbuncles,
furuncles, abscesses
2. It can bring about serious conditions like septicemia,
endocarditis, puerperal sepsis, osteomyelitis and pneumonia
3. It can cause food poisoning
4. It is a common cause of nosocomial infection

Pathogenic determinants:
1. Capsule – its presence has anti-phagacytic activities
2. Enzymes:
a. Coagulase – brings about clotting of plasma
b. Hyaluronidasa – also known as the spreading factor or the Duran-
Reynal factor
c. Staphylokinase – proteolytic enzymes with fibrinilytic activity
d. Nuclease – cleaves either the DNA or the RNA
e. Lipases – hydrolyzes fats and oils on the surfaces areas of the body
f. Penicillinase – acts on penicillin
3. Toxins:
a. Exotoxins – hemolysins:
Alphahemolysin – lyzes sheep’s and rabbits rbc but not man’s rbc
Beta hemolysin - lyzes sheep’s cells but not rabbit’s and man’s rbc
Delta hemolysin – lyzes human, sheep and rabbit rbc
b. Leukocidins – destroy exposed white blood cells and
polymorphonuclears
c. Enterotoxin – cause of food poisoning
d. Exfoliative toxin – epidermolytic causing destruction of epidermal cells
Straphylococcus epidermidis
Known in the old nomenclature as the Staphyloccus albus because it usally
produces white colonies. It is a normal inhabitant of the skin but can cause minor
conditions like stitch abscess and blepharitis. It is coagulase negative and does not
ferment mannitol.
Staphylococcus saprophyticus – rarely but can cause urinary tract among sexualloy
active women especially in Europe.
S. aureus S. epidermidis
1. pigment golden yellow porcelain white
2. Hemolysis hemolytic non-hemolytic
3. Catalase test + +
4. Coagulase test + +
5. Mannitol fermentation + +
Catalase test – 1 ml of 3% hydrogen peroxide is allowed to flow over the growing
colonies. Positive is when bubble of oxygen are produced.

Point to consider : RBC contain catalase so the test must not be performed
Directly on organism grown on BAP. Colonies maybe picked
up and test be done on a glass slide.
Coagulase test – either rabbit or human plasma can be used. Fresh plasma is
preferred. Both the slide and the test tube methods are available.
- 0.5 ml plasma is mixed with a largely colony of the organisms.
- positive results is clot formation in test tube and visible clumps in
slide method.
STREPTOCOCCI
The streptococci are a heterogeneous group of gram + spherical to avoid cells.
Cell division occur in parallel planes in adjacent cells resulting in pairs and in chains.
Member are facultative anaerobes. They are fermentative, catalese negative and
generally metabolically active.
Classification of Streptococci:
I. Based on physiological development according to Bergey
1. Hemolytic
2. Viridians
3. Lactic
4. Enterococci
5. Peptostroptococci – obligate anaerobe
II. Based on the hemolytic activities according to Smith and Brown
1. Alpha hemolytic - colonies are surrounded by greenish zone
- considered to be partial or incomplete hemolysis
2. Beta hemolytic - colonies are surrounded with a clear zone
- considered to be complete hemolysis
3. Gamma hemolytic - no change or hemolysis
III. Based on serologic identification according to Lancefield
Serological identification is possible because species differ in polysaccharide
(C-CHO) present in-between the cell wall and the cell membrane.
Streptococci are designated in letters A to O.
Important species:
1. Streptococcus pyogenes

2. Streptococcus agalactiae
3. Streptococcus faccalis
4. Streptococcus pneumonia
5. Streptococcus viridians
Streptococcus pyogenes - Group A streptococci
The organism is beta-hemolytic, non-motile, non-spore former with few species
provided with a capsule. It is associated with a wide range of disease like:
1. sore throat - pharyngitis, tonsilities
2. skin infection - pyoderma, impeti, cellulitis
3. scarlet fever
4. puerperal sepsis
5. post-streptococcal conditions: acute rheumatic fever (ARF)
acute glumeerulo-nephritis (AGN)
1. M-protein is a surface that enable the organism to resist phagocytosis
- can precipitate fibrinogen
- can clump platelets and leucocytes
- can inhibit migration of leucocytes
2. Toxins:
a.1. Hemolysins a. 1. Streptolysin O (SLG) – toxic for blood cells and
myocardial cells. Inactivated by 02
a. 2. Streptolysin S (SLS) – oxygen labile that can
produce hemolysis of blood cells
b. Eryhtrogenic toxin / Pyrogenic toxin - responsible for the rashes that
appear in scarlet fever.
3. Enzymes:
a. Nucleases: DNase - depolymerize the DNA
RNase - depolymerize the RNA
b. Streptokinase - digest fibrin and other protein
c. Hyaluronidase - splits hyaluronic acid
d. Diphosphopyridine nucleotidase - related to the ability of the
organism to kill white blood cells
Isolation: Specimens: Threat culture or tonsillar swabs
1. Perform a Gram staining procedure.
2. Gram positive cocci should be subjected to Catalase test.

N.B. Staphylococci are catalase +
Streptococci are catalase -
3. Inoculate catalase negative organism to blood agar plate.
B- hemolytic - Streptococci Groups A, B, C, D, & G
Alpha-hemolytic - Streptococcus pneumonia & S. viridans
4. To the beta-hemolytic organisms, perform the bacitracin test.
Positive - Group A
Negative - Groups, B, C, D, & G
Basitracin sensitivity test
Streptococcus pyogenes is sensitive to low concentration of bacitracin.
1. Select a colony from the culture to be tested.
2. Streak it evenly over the surface of a blood agar plate.
3. Place a bacitracin disk in the center of the area streaked.
4. Incubate overnight.
5. A zone of inhibition of any size indicates sensitivity.
Serological procedures to detect anti-streptococcal antibodies:
1. Anti-Streptolysin O Test/Titer (ASOT)
The test depends on the neutralization of the hemolytic action of
Streptolysin O reagent on erythrocytes by the antibodies present in the
patient’s serum.
2. Anti-deocyribonuclease-B test (AND – B)
This is another neutralization test in which the streptococcal enzyme
DNase B is used as the antigen. The antigen is added to the patient’s serum
and depolymerization of DNA will have to be observed.
3. In vive procedure - intradermal test (IDT) for scarlet fever
a. Susceptibility test: Dick’s test
Erythrogenic toxin is injected intradermally. The patient is susceptible
if at the site of injection will develop redness and edema within 18-24
hours,
b. Neutralization test: Schultz-Charlton test
At the site of rashes, anti-crythrogenic antitoxin is administered.
Fading of rashes, anti-crythrogenic antitoxin is administered. Fading of
rashes and eventual dis-appearance of the same will indicate that the anti-
toxin has neutralized the toxic activity of the pyrogenic toxin.

Streptococcus agalactiae - Group B Streptococci
This is a beta-hemolytic organism. It has been isolated from cases of neonatal
septicemia and congenital pneumonia. This has been associated with meningitis of the
neonates and the young ingants. The organism may be identified from other beta-
hemolytic streptococci by organism may ne identified from other beta-hemolytic
streptococci by the:
(1) Hippurate hydrolysis
a. A loopful of the organisms should ne transferred to a test tube with 0.4 ml
of 1% acqueous sodium hippurate.
b. Incubate for 2 hours at 320C
c. Add 0.2 ml of ninhydrin solution
d. Incubate at 370C for 20 minutes.
Hydrolysis of hippurate is indicated by deep purple color. Negative reactions
colorless.
(2) CAMP Test after (CHRITIE, ATKINS, MUNCH & PETERSON)
This uses beta-toxin producing Staphylococci.
a. Streak the beta-toxin producing staphylococci across the center of sheep
blood agar plate.
b. Streak the streptococci to be tested at right angles to the staphylococci
from the edge of steak across the uninoculated portion of the plate.
c. Incubate aerobically for 18 hours.
A positive CAMP test is indicated by an arrowhead-shaped zone of hemolysis
around the test organism where it about the staphylococci.
Enterococci – Group D Streptococci
These are usually found as normal constituent of the fecal flore. However, they
have been associated with cases of urinary tract infection, intra-abdominal abcess and
peritonitis. Identification is based on:
1. ability to grow in 1% methylene blue
2. ability to grow in 6.5% soduimchloride bile esculin agar.
3. Ability to grow 450C
N.B. There are other group D Streptococci but non-emtorococcal. The tolerance test to
6.5% NaCl can very well differentiate Group D Enterococci from non-Enterococcal
Group D.
Tolerance to 6.5% NaCl test

1. Inoculate a brain heart infusion (BHI) supplemented with 6.5% sodium chloride
with a colony of the organism
2. Incubate for 48 hours
3. Observe for turbidity, or toher evidence of growth.
Streptococcus viridians
The so called viridians group has been designated to a large member of alpha
reacting streptococci that resist classification by group-specific carbohydrates. This
heterogenous collection of streptococci are generally found in the mouth or upper
respiratory tract, in the gingivalcrevices or in the gastrointestinal tract, in the female
genital tract and occasionally in the skin. They are often mistaken as s. pneumonia.
The recognizedspecies include:
1. S. sanguis) hace been associated withdental caries and dental plaque
2. S. mutans)
3. S. mitis )
4. S. milleri ) unusual predilection to produce absxesses in
5. S. intermedius – MG) the tissues, such as the brain or the liver.
6. S. sanginosus )
N.B.S. sanguis is the most frequent single species that can cause bacterial
endocarditis.
Streptococcus pheeminiae
Also known as Diplococcus pneumonia since they typically occur in pairs although
they also occur in short chains. They pneumococci are characteristically encapsulated
and lancet-shaped. On blood agar plates they develop colonies surrounded alpha
hemolysis. Unlike other streptococci they require nitrogenous alcohol, the choline for
nutrition.
Medical Importance: most common cause of acute bacterial pneumonia
Extra-pulmona can cause middle our infection
sinusitis
meningitis
mastoiditis
Pathogenic determinants :
1. Polysaccharide capsule – with anti phagocytic effect
2. Pneumolysin 0 –with hemolytic activity
3. Neuraminidase – contributes to the invasiveness of the organism by favoring
Growth in the nasopharynx and in the mucous membrane of
the bronchial tree

To differentiate from Strptococcus viridians we can perform:
S. pneumonia S. viridans
1. Bile solubility + -
2. Optochin test + -
3. Quelling test + -
4. Inulin fermentation + -
5. Animal inoculation test + -
Bile solubility test
This is based on the presence in the pneumococci of an autolytic enzyme,
emidase that cleaves the bond between alanine and muramic acid in the peptidoglycan.
The amidase is activated by surface active agents such as bile or bile salts. The result
is dissolution of the organisms.
The test must be carried out at neutral pH. Using deoxycholate and live young
cells in saline suspension.
Optochin test
Streptococcus pneumonia is sensitive to optocnin which is ethyl hydrocuprein
hydrochloride.
(1) Streak half the surface of a blood agar plate with a colonyof the test organisms.
(2) Place an optochin disk in the center of the streaked area.
(3) Incubate overnight in the absence of increased C02.
(4) Zones of inhibition of least 15 mm represent highly presumptive evidence of
pneumococci.
Quellung test or the Neufeld-Quellung reaction or the capsular swelling
This is considered to be the most useful method in identifying the pneumococci. It
can be used directly for the identification of the organisms in sputum, spinal fluid,
exudates or culture.
(1) Mix the organisms with a loopful of anti-pneumecoccocal-antiserum and methylene
blue
(2) Examine immediately under the oil immersion lens.
Positive reactions is the capsule becomes more refractile and greatly swollen in
appearance.
Animal Inoculation
If there are no organisms from the spuctum specimen from a patient suspected of
pneumococcal pneumonia, the sputum can be injected intraperitoneallyinto a white
mouse. The mouse is higly sensitive to thecapsulated S, pneumonia and usually

succumbs to fatal infactions 16-40 hours after infection. The mouse effectively
eliminates other organisms in the sputum and pneumococci may be isolated in pure
culture from the heart blood of the animal.
Other Gram-positive Cocci
1. Gaffyka Netragena – characteristically arranged in packets of 4 or tetrads.
2. Sardine – Lutea – occurs in cubical packets of 8’s, 16’s, 32’s or more.
Also known as the gaprohytic – micrococci. Procedures bright yellow
pigments. A common contaminants in the laboratory.
NELSSERIA – GRAM – NEGATIVE COCCI
The organisms are usually seen in pairswithadjacent sides flattened, hence
known also as the coffee bean shaped organism. They are non-motile catalase positive
and sytochrome-oxidase positive. They ferment glucose. They are fastidious
organisms because in their growth, they require free iron and the presence of 2 – 8%
C02.
Media of Choice – Chocolate agar plate (CAP)
Transgrow – a transport medium to maintain viability of the organisms
during transportation
Thayer-Martin medium – very similar to the chocolate agar to which
antibiotics have been added.
The antibiotics are:
V – to inhibit growth of G+cocci
C –olisthemethate – to inhibit growth of G- org
- ystatin – to inhibit growth of fungi Trimethoprim
loctate is also added either to the Transgrpw or
the Thayrer – Martin to check swarming of the
colonies.
Species :
1. Neisseria gonorrheae – gonococci
causative agent of gonorrhea
2. Neisseria meaningitidis – meangicocci
causative agent of ipidemic meningitis
3. Neisseria flava)
Neisseria flavescenes ) so called non-pathogenic neisseria
Neisseria sicca ) they are part if the normal flora

Neisseria mucosa ) although they can occasionally cause
Neisseria lactamica ) disease.
NEISSERIA gonorrhea
Fresh isolates were found to be capsulated.
Small colony types are piliated
Sensitive ti drying and exposure to sunlight hence the necessity for a transport medium
(Transgrow)
Causative agent of gonorrhea, manifestations of which are:
1. Urethritis
2. Cervico-vaninitis
3. Rectal involvement
4. Phryngealo involment
5. Pelvic inflammatory disease (PID)
6. Opthalmia neonatorum – to prevent occurrence, give the crodes prophylaxis
which is instillation of 1% AgNO3 solution into the eyes of the newborn.
Identification
1. Stained smears of pus/secrations from urethra, vagina, cervix, prostate, rectal
mucosa and threat. Recovery of Gram-diplococci of the condition. When
intracellular, it is considered to be truly diagnostic while if extracellularly, only
presumptive.
2. Cultivation. Colonies are transparent, non-pigmented, non-hemolytic convex,
glistening and elevated.
3. Presumptive identification. Perform the Oxidase test. All Neosseria are oxidase +
Oxidase Test
1. Saturate a filter paper contained in a petri dish with either:
a. 0.5% tetramethyl-p-phenylenediamine HCL or
b. 1.0% para-amine-dimethylaniñene monoHCL
2. Pick a portion of the colony with a platinum dish and rub the colon on the will
paper.
Positive Results:
With 0.5% tetramethyl-p-phenylenediamine in 10 seconds, a deep purple color
willappear.
With 1.0% para-amine-dimethylaniñene monoHCL, stand for 15 minutes, colonies
will change to pink then darken to blank. If negative donot discard until after 1-2
hours.

4. Confirmatory test
a. Fluorescent Antibody Tagging/Test (FAT)
b. Degradation of Carbohydrates. The recommended base medium is Cystine
Trypticase agar (CTA)
Neisseria gonorrhea ferments only glucose with acid but no gas.
NEISSERIA meningitides
- another gram negative diplococcic
- fresh isolates demonstrate presence of capsule
- also piliated
- causative agent of meaning-coceal meningitis
- for identification:
1. carriers – nasopharyngeal, swab
2. cerobro-spinal fluid (CFS)
1. Gram staining
2. Cultivation
3. Quelling reaction with specific antisera
4. Carbohydrates degradation
N. gonorrhea N. meningitides
a. glucose + +
b. maltose - +
c. sucrose - -
d. lactose - -
e. fructose - -
BACILLI
GRAM-NEGATIVE group – The ENTEROBACTERIACEAE
They are enteric bacteria physiologically found in the colon, hence often
referred to also as coliform oraganisms. They are a group of heterogenous straight
rods that are morphologically similar to one another. Some are motile while other are
non-motile. If motile, the organism is provided with peritrichous flagella. They are
facultative a necrobes and non-spore former. All members are to nitrites but all are
oxdase negative.

Important tribes: Bergey’s Manual of Determinative Bacteriology
I. Escheriaceae – Escherichie
Edwarsiella
Citrobacter
Salmonella
Shigella
II. Klebsiellae – Klebsiella
Enterobacter
Hafnia
Serratia
III. Proteae - Proteus
Previdencia
IV. Yersiniae - Yersinia
V. Erwiniae - Erwinia
Medical Importance:
Pathplogically, all general are responsible for sporadic or epidemic diarrheal
disease, acute gall bladder infection, genitor-urinary infection, appendicitis, peritonitis,
septicemia and bacteremia.
1. Endotoxins – derived from the cell wall of the bacterial cell and are liberated when
bacterialyze. They can produce the following effects.
a. fever/pyrogenicity
b. tolerance
c. lethalshook from gastrointestinal hemorrhage
d. abortion
2. colicins – bacteriocins with antibiotic like substance resulting on the death or lysis of
the producing cell thus releasing the endotoxins.
PRIMARY differentiation of the members can be based on:
LACTOSE FERMENTATION: Rapid, Non-fermenters, slow.
1. Rapid fermenters produce colored colonies on bile lactose medium
- members are usually non-pathogenic
1. Escherichia
2. Enterovacter
3. Klebsiella
2. Slow-fermenters : in the past were referred to as the PARACOLON
1. Edwardsiella
2. Cirtrobacter

3. Hafnia
4. Serrabia
5. Providencia
6. Erwinia
3. Non-lactose fermenters – produce colorless colonies
- usually are pathogenic
1. Salmonella
2. Shigella
3. Proteus
There are umerous MEDIA useful in the detection of lactose formentation and to
mention a few, any of the following can be used:
1. EMB – Eosin-Methylene Blue
2. MacConkey Medium
3. ENDO Agar
4. RDS – Rusell’s Double Sugar
5. KIA – Kligler’s Iron Agar
6. TSI – Triple Sugar Iron
However, only reaction in the TSi will be further discussed. On TSI, degradation of
sugar and accompanying acid production are detected by the H indicator, phenol red
which changes its color from red orange to yellow. On alkalinization, it becomes deep
red. Thiosulate is reduced to hydrogen sulfide, which reacts with an iron salt to give
black iron sulfide.
TSI reaction Interpretation
1. acid butt; alkaline plant glucose fermented
2. acid butt; acid slant lactose and/or sucrose fermented
3. presence of gas bubbles/ cracks there was gas formation
4. blackening of butt production of H2S
5. media remained orange red one of the sugars was fermented
rapid lactose fermenter:
Escherichia
Type species: Escherichia coli or the colon bacillus
- predominantly present on the colon, hence thought to
be mainly non-pathogenic
- can become opportunist and can be:
1. EPEC – entero-pathogenic Escherichia coli
- associated with nursery outbreaks of diarrhea

2. ETEC – entero-toxigenic E. coli
- associated with traveller’s diarrhea
3. EIEC – entero-invasive E. coli
- associated with dysentery – like disease
Strains of E. coli
1. E. coli var communior – can ferment sucrose
2. E. coli var communis – does not ferment sucrose
3. E. aurescens – produce yellow orange pigment
Laboratory identification :
1. Stained smears
2. Cultivation on :
a. EMB – develops greenish metallic sheen
b. macConkey medium – produces pinkish colonies
c. XLD – Xylose Lysine Desoxycholate – produce yellow colonies
3. Biochemical Reaction :
a. TSI – acid slant over acid but with gas formation
b. IMVic reaction: + + ----
4. Serologic typing
IMVic test :
In-dole test – In the presence of oxygen, some bacteria are able to split
tryptophan into in-dole and alpha propionic acid. Release of in-dole
can be tested either by the Kocac’s or Ehrlich’s method.
Kovac’s method :
1. Inoculate a tryptophan broth.
2. Add approximately 0.5 mL of the Kovac’s reagent.
Results : + deep pink to red color
- yellow color of the reagent
Kovac’s reagent : - dimethylaminobenzaldehyde dissolve in amyl or isoamyl
alcohol with concentrated hydrochloric acid
ehrlicils method
(1) Add 1 mL of xylene to 48 hours culture. Shake well to allow the xylene
to rise to the surface.
(2) Add 0.5 mL of Ehrlich’s reagent slowly to the side of the tube I order to
layer the reagent in between the broth and the xylene.
Results : + red ring below the xylene layer.

Ehrlich’s reagent : p-dimethylaminobenzaldehyde in absolute alcohol iwht
concentrate HCl
m-ethyl Red Test – some Gram – bacilli are able to produce a high degree of acidity
which is detected by the methyl red indicator. The indicator can detect a
pH range of 4, 5 to 6
(1) Inoculate MR-VP medium with the organism.
(2) Incubate at 300C for 5 days
(3) Add0.2 mL of the methyl red reagent per 5 mL of culture.
Results : + bright red color
- yellow or orange color
Vi-Voges – Proskauer Test – some bacteria are able to produce acetoin from glucose.
In an alkaline pH. acetoin is oxidized to acetyl-methyl carbinol
(1) Inoculate 5 mL of Mr. VP medium with the organism
(2) Incubate at 350C for 48 hours
(3) Add 0.6mL of 5% alpha-napthel in absolute alcohol. Mix well
(4) Add 2.0 mL of 40% KOH containing 0.5% creatine. Shake well
Result : + production of red color on 5 minutes.
C-itrate Utilization Test – Simon’s citrate agar incorporates citrate as the sole source of
carbon in the medium, which when metabolized, leads to the alkalinization of the
medium. It is indicated by a change in the color of pH indicator bromthymel blue from
green to blue.
KLEBSIELLA type species is Klebsiella pneumonia –
- also known as the Friedlander’s bacilli or the Bacilli
mucosus capsulatus
- no-motile Gram – capsulated rods.
- Has been associated with :
a. Severe enteritis
b. Pneumonia
c. Septicemia
d. Meningitis
1. Stained smears
2. Cultivation in: EMB, MacCcokey, XLD

In these media, organism develop large mucoid colonies that have a tendency to
coalesce. Colonies exhibit positive String test
3. Biochemical;
a. TSI – acid slant over bitt with gas
b. IMVIC Reaction - - + +
c. Ggrows in KCN medium
d. Liquefies gelatin
e. H lyzes urea
Other Klebsiellae :
1. Klebsiella oxytoca – sa – k. pneumonia but indol +
2. Klebsiella ozaenae – implicated as the cause of shonic astrophic rhinitis
3. Klebsiella rhinoscleromatis – produces a granulomatous destruction of nose
ENTEROBACTER type Enterobacteraerogenes
- Formerly known as Aerobacter
- Often confused with Klebsiella
- Motile
- TSI reaction – acid slant over butt with gas
- IMViC reaction - - + +
- Grows in KCN medium
- Most are urease +
Klebsiella pneumonia Enterbacter aerogenes
IMVic reaction - - + + - - + +
Motility non – motile motile
Ornithine decarboxylase - +
Ornithune decarboxylase test : The broth contains orthine as a reactant and promeresol
pruple as a pH indicator. At pH below 5,6, the pH indicator is yellow. Ornithine
decarboxylase positive organisms cause the pH to rise. As a result of the breakdown of
ornithine, the culture medium becomes violet.
Non-Lactose Fermenters :
SALMONELLAE Gram – aerobic rods, non-spore-formers, with perotrichous
flagella with the exeption of Salmonella pollurum and Salmonella gallinarum
Species of Medical Importance :
1. Salmonella typhi also known as Eborth’s bacilli

Salmonella typhosa
Eberthella typhosa
2. Salmonella Paratyphi A –
3. Salmonella “ B – Salmonella schotmuelleri
4. Salmonella “ C – Salmonella hirschfeildi
5. Salmonella cholerasuic – type species
6. Salmonella typhimurium
7. Salmonella enteritidis – Gartner’s bacilli
Pathology : These various organisms are causing a group of infection called
SALMONELLOSIS which includes:
1. Enteric fevers associated with Salmonella typhi and Salminella Paratyphi A
2. Septicemia associated with : Salmonella Paratyphi B
Salmonella Paratyphi C
Salmonella typhimurium
Salmonella cholerasuis
3. Gastroenteritis associated with :
Salmonella enteritidis
Salmonella typhimurium
Salmonella Paratyphi B & C
Typhoid fever – a form of entire fever which is an acute infectious disease characterized
by continued fever, inflammation of the intestine, formation of intestinal
ulcers with characteristic rose in the abdomen.
Antigen produced in Salmonellosis :
1. O Antigen – somatics
2. H antigen – flaggelar
3. Vi antigen – capsular antigen to stand virulence
First week of infection – organism may be isolated from blood
Second week and on – organism may be isolated from steel and urine
Third Week and on – antibodies appear in the serum
N.B. Drainage from the bilinary tract is useful in the identification of carriers.
Diagnosis is made possible through :
(1) Stained smears
(2) Cultivation : EMB, macConkey & Salmonella – Shigella agar (SSA) organisms
develop colorless colonies
XLD)
organisms develop black colonies

BSA)
BSA (Bismuth Sulfide Agae) – considered to be the best
medium for the isolation of Salmonella typhi
Brilliant Green Agar ) BGA) – organisms develop slightly pick colonies to
white opoque colonies surrounded by brilliant red color
- this is highly recommended for the isolation of all Salmonella except
for S. typhi
(3) Biochemical :
a. TSI reaction : Salmonella typhi – alkaline slant over acid butt with small
amount of H2S
Other salmonella – K/A + H2S
b. IMVic reaction - + - +
(4) Serotyping with known antisera (commercially available)
(5) Serology : Widal test using two-fold dilutions of the patients serum interacted with 0
antigen titer of 1:160 indicates active infection
H antigen – indicates past infection
Or past vacation
Vi antigen – carrier state
N.B. Specimen is usually placed first in an enrichment media before inoculation in the
media mentioned above. Enrichment media can either be the Selenite F medium of
the tetrathinate broth.
SHIGELLAE another non-lactose fermenter that are lose invasive than the Salmonella
o Gram – slender, non-encapsulated and non-motile
Species of medical importance :
According to mannitol fermentation :
I. Non-Mannitol fermenter
1. Shigella dysenteriae – Shiga bacilli
- Type species
II. Mannitol fermentor :
A. Lactose non-fermentor 1. Shigella flexneri
Strong bacilli
2. Shigella boydii – Boyd’s bacilli
B. Late lactose fermenter – Shigella sonnei/ SonneDuval bacilli

Pathology : All cause bacillary dysentery which is characterized by sudden onset of
abdominal cramps, diarrhea and fever after an incubation period of 1-4 days. Mucus
and blood there are plenty of pus cells.
1. Stained smear
2. Cultivation : EMB, SSA, MacConkey – colorless colonies
XLD – pinkish to raddish colonies
Heaktoen Enteric Agar (HEA) – green to blue green colonies
3. Biochemical :
a. TSI – alkaline slant ever acid butt
b. IMVic reaction – V + - -
4. Serotyping with known antisera
PROTEUS are actively motile Gram – pleomorphic rods
- All are urease positive
- All are phenylalnine deaminase positive (PAD)
- Exhibit Diene’s phenomenon – different stains of Proteus when inoculated
separately in the same culture plate, will exhibit swarming but will not mix,
instead will leave a demarcation line in between them.
- exhibit swarming phenomenon on non-selective medium where organism will
produce a thin transluescent sheet of growth. Can be prevented by incorporating
into the media anyone of the following substances :
1. sodium azide
2. chloral hydrate
3. phenyl-ethyl alcohol
- produces powerful urease that hydrolyzes urea into ammonia and CO2 raising the
pH of the media
species : Proteus vulgaris Proteus mirabitis
TSI Alkaline slant over acid with plenty of H2S
IMVic + + - V - + V V
Proteus vulgaris have the same antigenic structure as the Rickettsian, so that O
antigens coming from non-motile Proteus are used as antigens in the Weil – Felix test
detect antibodies in Rickettsial infections. These are : OX K, OX 19, OX 2.
Phenylalanine Deaminase (PAD)
(1) inoculate a phenylalanine agar slant.
(2) Incubate the culture at 350C for 18 – 24 hours.

(3) Disponse 4-5 drops of 10% ferric chloride over the growth.
Positive : a dark green color develops on the surface and in the fluid
Negative : yellowish color of the reagent
OTHER ENTEROBACTERIACEASE MEMBERS :
Edwardseilla : type species is Edwardsiellatardia
Motile
TSI : K/Ag alkaline slant over acid butt with H2S
IMVic : + + - -
In man, Edwardsiella infections is rare but the organism has been isolated from
wound infections, sepsis and even meningitis. It has been associated also with
gastreenteristis. Because of H2S production, some would mistake this for Salmonella.
CitronacteriArizona group
Both generation are closely related to Salmonella.
Citronobacter type species Citrobacterfroudii
- morphologically appears similar to Escherichia coli
- biochemically resembles Salmonella
- TSI : acid slant over butt with H2S
- IMVic : - + - +
- Urease +
- Grows in KCN
Arizona type species Arizona Hinshawii
- are incidental pathogen man since it is common among birds and
reptiles
- motile
- late lactose fermenter
- TSI : acid slant over butt with H2S
- IMVic : - + - +
- Utilizes malonate
Malonate utilization test :
(1) Inoculate a malonate broth with test organism.
(2) Incubate for 18-24 hours at 370C.
Degradation of malonate result in an alkalinization of the medium which is indicated
by a change in color of the pH indicator bromthyol blue from green to blur.
Positive : culture medium turns blue
Negative : culture medium remains green

KCN (Potassium cyanide) test
The growth of some microorganism is inhibited by very low concentration of KCN.
Less sensitive microorganisms can however grow in this media as exhibited by the
presence of turbidity.
Serratia - thought to be saprophythes but a present are currently emerging as a very
common cause of nosocomial infections.
- Members : Serratia marcescene
Serratia liquefactions
Serratia rubidasa
- TSI : K/A alkaline slant over acid butt
- IMVic : - - + +
- some species produce pink to red pirgments called prodigiesin
- specialization can be based on :
S. marcescons S.liquefasience S. rubidaea
Ornithine
Decarboxylase + + -
Arbinose
Fermentation - + +
DNAse + + +
- production of the DNAse makes them different from the other members of the
Enterbacteriaccae.
MORGANELLA and PROVIDENCIA
Both are closely related to the Preteus group, because both claborate urease
and both produce indole. They however do not produce hydrogen sulfide.
Morganella morganii TSI : K/AG alkaline slant ever acid butt
IMVic : + + - -
Providencia rettgeri TSI : alkaline slant over acid butt
IMVic : + + - +
Erwinia not medically important since they are only pathogenic to plants
Yersinia

One major change in the taxonomy of the enteric organisms is the inclusion of
Yersinia in Enterobacteriaceae. All the three species under the Genus Yersinin
primarily pathogenic to animals but can also produce disease in man.
Yersinia postis also known as the Plague bacillus
- gram negative, non motile
- exhibits marked bipolar staining when stained with Wayson stain,
hence also as the safety pin bacilli
- causative agent of plaque, clinical forms of which are:
a) bubonic – resulting in the inflammation of lymphatic glands
called buboes. The condition will lead to necrosis.
b) Pneumonic – type of bronchopneumonia that is hemorrhagic. It
is airbone in transmission.
c) Septicemic – infection is already in the blood steam.
- Plague is also known as the Black Death.
1. Stained smear
2. Cultivation : MacConkey
Desoxychocolate agar – colonies are red
BAP – non-hemolysis
In brothcultures, may form surface growth pellicle termed as stalactite
streamers.
3. Biochemical : TSI – alkaline slant over acid butt
IMVic - - + - -
Yersinia pseudotuberculosis )
Yersinia enterocolition ) both are associated in man with gastrointestinal
disease and involment of the mesenteric lymphaties
calles yersinosis.
Both are motile
Both are urease
Y. tuberculosis produces hydrogen sulfide
Y. centerocalation break-down-ormithine
For the identification of the members of the Enterobacteriaceae, there are now available
kits defined as a serious of miniaturized or standardized tests are commercially
available

(1) A single colony is picked and inoculated into the test system
(2) Samples are incubated, read and the result are used in the identification.
This approach is similar to the conventional tube method with the main difference being
in the miniaturization, the number to tests available and the method of analyzing results.
For information alone, the following are the available kjits that have been proven to be
useful in the laboratory idenfitification :
1. Enteritube make use of the conventional media. It can perform 15 tests
at a time.
2. API (Analytab Products Inc.) allows 21 biochemical test with the use of
dried substrate.
3. Inolex Enterix allows 20 biochemical tests with the use also of dried
substrate.
4. Minitec and Pathotec allows the microbiologists to choose the
biochemical test.
5. R/B system uses also conventional media. It allows 14 biochemical test
6. AIM 4 system (Axford International) is based also on conventional media
7. Micro-ID system allows 15 biochemical test and results are read
immediately after 4 hours incubation. It used fried substrate.
8. Micro-Media quad Panel utilizes 20 standard biochemical test in small
volumes.
VIBRIONACEAE
The members of this grow on media used for the isolation o9f bthe enteric coliforms
and therefore could be easily mistaken as members of Enterobacteriaceae.
All members are Gram negative.
All members are oxidase positive.
They are motile by means of popular flagella.
Species of media impprtance:
1. Vibric cholerae
2. Vibrio parahemolyticus
Vibrio cholerae
- short rods but on primary isolation appear comma-shaped hence also known as
the Kommabacillus. On stained specimens, they appear lying parallel to one
another that they are commonly described as “fish in the tream”

- with biotype V. El Tor. It could be differentiated from the biotype cholerae
because the former is positive to Voges – Proskauer test, can agglutinate the
chiken erythrocytes and is not sensitive to polymyrin B.
- causative agent to cholera which is acute infectious epidemic disease
characterized by vomiting and passing out of rice watery stools with abdominal
cramps.
- Pathogenic determinants :
1. Enterotoxins also known as choleragen. It is responsible for the
hypersection of the intestinal mucosa.
2. Endotoxin which is responsible for thee denudation of the intestinal
epthelim.
3. Motility of the organism has something to do with ability of the V.
cholerae to penetrate the intestinal mucus and get attached to the
micro-villi.
1. Hanging drop preparation of stool/vomitus. Daring motility can be observed.
2. Dark-field microscopy will also useful in the observation of motility.
3. Cultivation :
It is important to remember that V. cholerae is highly susceptible to drying and
acidic conditions. Top prevent dessication, specimens e.g. vomitus, stool or rectal
swab should be placed in an transport medium like Amies medium, Cary Blair or the
Stuart transport medium. The acidic conditions can sterilize the organism, so pH
much be properly checked. The organism survive at a pH of 8.5 – 9.5. from the
tranportmedia, the specimen can be transferred to any of the following media:
1. Alkaline peptone water
2. Dieudonne’s medium
3. Tellurite Taurochocolate Gelatin Agar (TTGA)
On this medium. Growing colonies are surrounded small cloudy zone of
hydrolyzed gelatin.
4. Thiosulfate citrate bile salts sucrose (TCBS)
It has a pH 8.6 Growing colonies develop small yellow colonies
with opaque and
5. Animal Inoculation test o to observe the Pfeiffer’s phenomenon
v. cholerae are inoculated into an immunized guinea pigs.
Lysis of the organisms will occur.
6. Serology
a. Slide agglutinatin test

b. Fluorescent antibody test
c. Immobilization test
Vibric cholerae sere group O has three sere group O has three sere
types namely :
1. Inaba type
2. Hikajima type
3. Ogawa type
Other vibries :
Vibric parahaemolyticus is another human pathogen causing astroenteritis and
food poisoning associated with the consumption of contaminated sea foods. It survives
in the presence of high salt concentration hence designated as halophilic.
Viric fetus – now moved to another genus Campylobacter.
It is a comma-shaped organism, which is highly motile with unipolar or
binopolar flaggelum.
Proophylaxis : Administration of a triple vaccine, CDT.
C-holera - Vibric cholerae infection
D-ysenteriae - Shigella infection
T-yphoid - Salmonella infection
NON-FERMENTATIVE Gram-Negative Bacilli
There are organisms whose energy from carbohydrates is derived by oxidative
rather than fermentative metabolism. The acid produced by the oxidative pathway is
less than that produced by other organism using a fermentative pathway. To test for
this type of carbohydrates utilization, the organism must be tested in a special mediu
like the O-F (Oxidative-Fermentative) of Hugh and Leifson.
To the medium is added a carbohydrate. The degradation of this carbohydrate to
acid is indicated by the pH indicator brothymol blue which changes its color to yellow.
The degradation is allowed to take place while the medium is exposed to air or under
exclusion of air. To exclude air one tube will have to be provided with a paraffin seal.
A yellow coloration in both the open and paraffin sealed tubes signifies fermentative
degradation while yellow coloration of the open tube alone indicates that the
carbohydrates is brokendown by oxidation. Oxidative breakdown take place at or close

to the surface. Fermentative breakdown occurs both at the surface and throughout the
butt.
Pseudomonas sp. Also known as the water bug organism
- theyresemble the enteric organism
- they grow well on enteric oraganism
- oxidase +
- motile with popular flagella
1. Pseudomonasaeruginosa
- non-encapsulated Gram negative ts woth mono trichoud flagella
- has been isolated from cases of :
a. wound infection
b. cystic fibrosis
c. otitis media
d. meningitis
e. urinary tract infection (UTI)
f. pneumonia
g. secondary burn infection
h. many nosocomial conditions.
Laboratory identification
1. Stained
2. Cultivation :
- grows in many media. It can survive well on media for enteric
organisms.
- Can tolerate alkaline environment hence it was found to survive also in
media vibric
- On BAP, colonies are beta-nemolytis producing large flat colonies with
ground glass appearance
- Produced diffusile blue green pigment called the pycyanin and greenish
yellow pigment called flouresceein
- Produces characteristic odor that id described as sweet aroatic hence
also called fruiy or grape-like odor.
- On O-F medium open produces a positive reaction
- Bedside diagnosis is possible through the use of the Wood’s light
because of the Flourescein pigment
2 Pseudomonasmallei (formerly under Actinobacillus)
- also known as the Glander’s bacillus
- non-motile, non-sporeformer, non-capsulated

- oxidase negative
- causative agent of glanders which is a common disease of horses.
Form the horses, the infection ma be transmittedto man. Cutuneous
involvement where there is thickening of the suoerdicial lymphatics is
called Fercy.
- Laboratory diagnosis includes animal inoculation utilizing male guinea
pids. The animal is inoculated intra-peritioneally with the organism.
Then observe for the Strauss reaction which is swelling of the seotum
after about 4 days and eventual necrosis after 1-3 weeks.
3. Pseudomonas pseudomallei – thecausative agent of meloidosis in rodents. It is
non-motile.
4. Pseudomonas maltophila – has been frequently isolated from the oropharynax and
sputum of normal adults.
- Organism is motile with lophotrichous flagella
- Produces uellow-tan pigments
- Develops an ammoniacal odor
Alcaligenes faecalis
- non-fermentative but also non-oxidative so when grown can O-F medium both the
close and the open will be negative
- short Gram negative rods that are sluggishly motile weakly oxidae +
- catalase +
- on MacConkey agar produves colorless colonies
acinotobacter group
This genus is divided into two groups:
A. Those that fermat glucose include :
1. Herella vaginicola
2. Bacteriumanitratum
3. Achromobacter anitratum
4. Acinetobacter anitratum
B. Those that do not fermet glucose include :
1. Mima polymorpha
2. Acinetobacter iwoffi
3. Achromobacter hemolyticus
The type species is Acinetobacter calcoaccticus which is :
- oxidase negative

- catalase positive
- non-motile
- Gram negative diplobacilli
Moraxella group
- Members are oxidase +, non-motile organisms
- Non-fermentative, non-oxidative
1. Moraxella lacunata – formerly known as the Hemophilus duplex
- also known as the Morax-Axenfeld bacilli
- isolated from cases of blepharo-cnjuctivities
2. Moraxella osloensis - commonly confused as Neisseria gonorrhea
- Gram negative coccobacilli
- Oxidase + -
- To differentiate from Neisseria, using the CTA medium,
NEisseria will be able to ferment the medium but M.
osloensis will not
Flavabacterium group type species : Flavobacterium meningosepyicum
- found to cause epidemic of meningitis among the newborns
- oxidase +, catalase +, gelatinate +
- non-motile
- develops cream piemet
- oxidizes glucose
SMALL GRAM-NEGATIVE BACILLI
This group of organisms generally requires enrichment for growth.
Haemophilus group
Members of the group are small, Gram negative aerobic, non-motile
non-spooreforming organisms. They affect the respiratory tract, the meninges,
conjunctive, and the genitor-urinary tract. The different species can be differebtiated on
the basis cultural requirements, the X and the V factors, the carbon dioxide need and
the presence of hemolysis.
Factor X – heat stable substance associated with hemoglobin
Factor V – heat stable substance, which is also known as the co-
enzyme I or the nicotinamide adenine dinu-cleotide (NAD). Factor V is
supplied by the blood but it can also be provided by yeast, potato
extract and certain organism like the staphylococcus, the Meissers and

the pneumococci.
Species Site of Infection X V Hemolysis Capsule
1. Hemophilus influence respiratory
Tract, blood, meninges + + - +
2. H. segyptus conjunctive + + - -
3. H. haemolyticus respiratory + + + -
4. H. parahemolyticus respiratory - + + -
5. H. parainfluenzae respiratory - + - +
6. H. ducreyi genitalia + - - -
Hemophilus influenzae
- alsp known as the Pfeiffer’s bacilli
- a secondary invader in influenza
- common cause of meningitis and atitis media
- Exhibits satellite phenomenon/satellitism
When the organisms are grown together with the organism that can
provide the V factor, growing colonies of H. influence are biggerand more
luxuriant.
- Grow best on Chocolate agar with incorporated Bacitracin Colonies are
small clear referred to as dewfrop-colonies which exhibit mousy odor.
- Can also cultivated in : Levinthal agar
Filde’s enrichment
- Can be identified by the :
1. bile solubility test
2. Quelling capsular swelling test
Hemophilus aegyptius - also known as the Kock-Weeks bacilli
- Associated with communicable conjunctivitis commonly calles
“Pinkeye” disease
Hemophilus ducreyi - also known as the Ducrey’s bacilli or chaneroid bacilli
- Causative agent of soft chancre or chaneroid or uncus molle.
It is a venerally transmitted disease characterized by
ulceration in the genetals.

- From smearsof the gential ulcers, organisms appears as
Gram negative rods in long stranic thus often referred to as
“School of red fish”
- Best grown in fresh rabbit or hseep blood
Bordetella group
The members are strict aerobes with absolute requirement for nicotinic acid.
They are associated with respiratory infections.
Bordetella pertussis – also known as the Bordet-Gengou bacilli
- causativeagent of pertussis or whooping caugh. The
condition usually start with a catarrhal condition similar to
ordinary colds. Then athe patient will suffer from paroxysmal
cough that ends with a whoop.
1. cultivation in : Bordet-Gengon medium made up of :
1. potato extra.t
2. blood about 50%
3. glycerol
Growing colonies have the appearance of drops of mercury or are sometimes
described as pearl like surrounded by a zone of hemolysis
- can also be grown on Joneskendrick Cahrcoal agar
N.B. Specimen collection can either be by :
a. nesopharygeal swab
b. cough plate method – a plate of the culture – medium is opened about 4 – 5
inches way from the face of a patient during the violent cough attack
bordetellaparapertussis )
) both can cause mild forms of whooping
Bordetella bronchiseptica ) cough
B. pertussis B. perapertussis B. bronchiseptica
1. Motility - - +
2. NO3 reduction - - +

3. Hemolysis + + +
4. Urease production - + +
5. Citrate utilization - + +
Francisella type species is francisella tularensis
- It is a poorly staining Gram negative coccobacillus.
- non-motile
- exhibits bipolar staining
- rarely encapsulated
- extremely pleomorphic
- causative agent of tularemia someyimes known as the rabbit fever. The
condition is characterized by a focal ulcer at the site of entry
accompanied by enlargement of the regional lymph nodes.
- Can be transmitted by ticks or fleas but can be acquired by direct
handling of infected animals.
Laboratory Identification :
1. Cultivation – culture medium is special and antibiotics must be added to prevent
overgrowth of other organisms.
- can be grownin cystine glucose blood
agar (CGBA). The organism do not exhibit true hemolysis but may
produce greening directly under the colonies.
2. Serology : Flourescent antibody test (FAT)
Agglutination test
Brucelle group
The members of this group are strict aerobes. They are small non-motile rods that
take Gram counterstain poorly. For growth they have complex nutritive requirement.
Generally they:
- reduce nitrates to nitrites
- are catalase +
- can hydrolyze urea
- produce H2S
- are associated with undulant fever/ Malta fever/ Gibraltar fever/
Mediterranean fever.
Curcently recognized species are :
1. Brucellaabortus

2. Brucellamelitensis
3. Brucellasuis
4. Brucellaneotamae
5. Brucellaovis
6. Brucellacanis
Brucellosis is primarily a desease of animals but from the infected animals the
organisms may be transmitted to man through:
1. contact with infected tissues and secretion of the animal
2. ingestion of contaminated/infected milk and dairy products
3. inhalation of airbone organisms
Species identification is based on various physiologic needs :
C02 H2S G R O W T H
need production Basic fuchsin : Thionine
1. B. melitensis - - + : +
2. B. abortus + + for 4 days + : -
3. B. suis - + for 5 days - : +
4. B. neotamae - + - : +
5. B. ovis + - + : +
6. B. canis - - - : +
Test for milk : Abortus Bang Ringprobe Test (ABR)
(1) Milk is mixed with hometoxylin stained organisms.
(2) Incubate at 370C for 1 hour
(3) Observe for agglutination which will take place.
Positive reaction : The stained bacteria will be carried to the surface with fat
globules.
Pasteurella group
The members of this group are small, non-motile Gram negative that exhibit
bipolar staining. There are four known species but the major human pathogen is :
Pasteurellamultocida
- non hemolytic but the choice for cultivation is a medium with hematin
- facultative anaerobe
- catalase +
- oxides +
- produces indole from tryptophan

- ferments mannitol
- produces sever gastreo-enteritis in fowls, birds and other domestic
animals but from these animals, the infection may be transmitted to
man.
Calyatobacterium has only one species the C. granulamatis
- also known as the Donovan body
- Gram negative, pleomorphic, non-motile
- usually heavily encapsulated
- exhibits bipolar condensation of chromatin giving rise or its safety pin
appearance
- causative agent of granuloma unguinale or denovasis which is
characterized by ulcerating granulomatous lesions usually in the
unguinal region. This s a chronic venereal disease.
Direct examination of biopsy material or granulation tissues from the skin lesion.
After air drying, the sample is stained with either the Wright’s or the Giemsa stain.
Microscopic examination of the stained mears should reveal intracellularDonovan
bodies inside the large mononuclear cells. The intracellular organisms appear as small,
straight or dumbbell curved rods stained dark blue and surrounded by a pinkish
capsule.
N.B. If only extracellular organisms are observed, repeat the smear and stain produce
to confirm the presence of typical intracellular – organisms.
MISCELANEOUG Gram Negative Bacilli
1. bacteroides fragilis
non spore forming anaerobic organism. They are quite
pleomorphic with vacuoles and swelling. It is the mostcommonly
isolated anaerobe from various infection. Growth of B. Fragilis is
stimulated by bile
2. fusobacterium nucleatum formerly called F. fusiforme. This is another Gram
negative anaerobic rod with pointed ends. They are non-motile.
On stained smears, they look like scattered wheat straw or very
long thin filaments. It is present in the normal flora of the mouth
and it is an important disease agent of oral infections, lung
abscess and other pleura-pulmonary conditions.

3. Bartonella bacillifornis parasitizes blood. They are small, gram negative,
polymorphic, motile bacteria. They posses ten terminal flagella. It
is transmitted by the Philebotomus fly or the sand fly. It can cause
two entirely different conditions :
1. Oroya fever – fatal hemolytic anemia
2. Verruga peruana – wart – like disease It
can be demonstrated from Giemsa-
stained blood smears.
4. Eikenella corrodens was formerly placed under Bacteroides but was found to be
different from te Bacteroides. It is a member of normal flora of the
mouth and the upper respiratory tract as well as the gastro-
intestinal tract. It has been isolated from infections of tissues
adjacent to their normal flora. It is usally associated with
streptococcal infections.
They grow well on media with blood and colonies show
pitting or corroding of the agar. They grow very slowly. The
organism is oxidase + but catalase -. It is positive to lysine and
ornithine decarboxylase.
5. Chromobacterium – violaccum – a rare pathogen that causes overwhelming
septicemia abcesses. It is motile with, one pola flagellum and 1 –
4 lateral flagella. It produces a violet indole-pyrrole pigment called
violecein. The organism is characteristically very sensitive to
hydrogen peroxide. The natural habitat of the organism I soil and
may also be present in natural water sources. It is believed that the
portal of entry is probably a skin lesion.
6. Gardnerella vaginalis formerly placed under the Hemophilus group then was moved
to Coryneabacteria. The organisms is a pleomerphic Gram
negative rod that can cause non-specific vaginitis. It can be
identified from Gram-stained vaginal discharge by the presence of
“clue Cells”, which are epithelial cells with plenty of small gram
negative bacilli. They are catalase and oxidase negative where
acetic acid is the major end product of fermentation.
7. Legiobekka pneumophila – Gram negative rod that requires enriched media for
growth. It is weakly oxidase + but strongly catalse +. It produces
a soluble pigment that results in the brown coloration of the
Feeley-Gorman (F-G) medium. It is associated with the
Legionnaires’ pneumonia.
It is also satisfactorily frown on charcoal yeast extract
(CYE) agar. Colonies look like ground glass with oblique light.

A direct fluorescent antibody test can also identify the
organism.
8. Actinobacillus ligieresii I is facultative anaerobe, non-motile fastidious Gram
negative rod. There is a tendency towards bipolarstaining. Sulfur
granules have been observed. They grow on peptome media
enriched with serum or blood. The organism is a common
pathogen of cattle and sheep but can be transmitted to man.
9. Cardiobacterium hominis is also facultative anaerobic, non-motile and fastidious. It’s
natural habitat is the upper respiratory tract but when it enters the
blood stream it will reach the heart and can cause endocarditis.
They grow on BAP but they are non-hemolytic.
GRAM POSITIVE BACILLI
Corynebacteria group
All members are Gram positive rods
All members possess irregular swellings on one end giving them
the so called “beaded appearance” or the club-shaped
appearance
All members develop the metachromatic granules
All members are catalese +
Corynebacteria diphtheria also known as the Klebs-Loeffler’s bacilli
-organism is non-motile non-encapsulated and non-spore former
-with irregular staining metachromatic granules called the Babes
Ernst granules
-pleomorphic that are arrange in palisades or at sharp angles with
one another giving the Chinese character appearance
-causative agent diphtheria
diphtheria is a toxigenic disease. Clinically the infection may be:
1. pharyngeal – classic form of this development of a local pseudo-
membrane covering the threat
2. cutaneous
-lysogenic strains of D. diphtheria which have rophage
with the “tox” gene produce the potent exotoxin
-toxigenic and non-toxogenic strains however can cause
diphtheria

-toxin produced by the organisms is absorbed
systematically and can damage distant organs like the
heart. Liver, kidneys and the nervous systems.
Specimens: throat and nasopharyngeal swobs
1. Stained smears – in the study of stained smears taken directly from the throat,
always consider the diphtheroids.
Diptheroids are diphtheria like organisms that are normally present in
the throat, thus very often can generally shorter and with less metahromatic
granules. They are non-pathogenic to man.
2. Cultivation on: Tinsdale medium
Pai’s a coagulated egg medium
Loeffler’s serum slant – this encourage pleomorphism. Colonies
have the “poached egg appearance”
cystine Tellurite Blood agar (CTBA)
with the use of tellurite, hydrogen sulfide is produced thus
developing a distinctive black zone of growth.
The cultural characteristic of the C. diphtheria were used as the basis of the
biotype classification into:
Corynebacterium Characteristics on Hemolysis Starch
Diphtheria CTBA on BAP & glycogen
Hydrolysis
(1) var gravis large, flat, dark gray - +
irregularly striated giving
rise to daisy head colonies
(2) var mitis small convex glossy black + -
colonies
(3) var intermedius very small, flat, dry - -
gray or black colonies
3. Toxigenicity test : also referred to as test for virulence
In vivo test – animal inoculation
Materials : 2 guinea pigs
broth culture of the test organisms diphtheria antitoxin
(1) The control animal is injected intraperitoneally with 250 units of antitoxin.
(2) Two hours later, the two animals will both be injected subcutaneously with 4
ml of broth culture of the test organism.

Results : If the inoculums injected to the test animals is toxigenic, the animal
usually dies within 24 – 06 hours.
The control animal is not affected.
b. In vitro test – also known as the Elektest
The testis based on a precipitation reaction occurring between
diphtheria toxin produced by the test culture and the diphtheria antitoxin in
the medium.
4. Test for susceptibility – the Schick’s test
Test arm Control arm
A 0.1 ml of purified diphtheris A 0.1 ml of inactivated diphtheria
toxin is injected on the volar surface toxoid is injection on the volar
surface
The site of injection are examined at 24 and 48 hours in 24 hours and between the
third and fourth days.
Results : Site of toxin injection begins to redden in 24 hours and increases and
reaches a maximum size in about 1 week, when it will swell and will
become tender. There is usually a small, dark red central zone that
gradually turns brown & leaves a pigmented area. (positive)
Site of toxoid injection shows no reaction. (negative)
Interpretation : If a positive reaction occurs, the individual does not have enough
antibodies to neutralize the toxin and is therefore susceptible.
OTHER Corynebacteria :
1. Corynebacterium ulcerans appear similar to C. diphtheria when grown on tellurite
media. Commonly found in the noses and treats of horses and in humans,
the organism cayse a form of tonsillitis. It can be differentiated from C.
diphtheria by the following :
C. dophtheria C. ulcerans
(1) hydrolysis of urea - +
(2) Reduction of N03 + -
(3) Acid from trehalose - +
(4) Liquefaction of gelatin - +
2. Corynebacterium ovis also known as C. pseudotuberculosis
It causes ulcerative lymphangitis, horse, abscess and other purulent
infections in sheep, horse, cattle and in man.

3. Corynabacterium minutiessium – causative agent of erythrasma, characterized by
the presence of scaly plaques which fluoresces enriched with bovine fetal serum,
producing colonies that display the coral rod to orange fluorescence similar to the
skin lesions. When grown on BAP, no characteristic florescence however occurs.
MYCOBACTERIA Group
This is a group of strictly aerobic organisms. The most distinctive properly of this
group is their characteristic staining. They stain with difficult, but once stained they are
resistant to decolorization with acid-alcohol hence referred to as acid – fast.
Mycobacterium tuberculosis –
- also known as the Koch’s bacilli
- acid-fast, non-motile, sporeformer, non-encapsulated
- weakly Gram positive
- with human and bovine strains
The bovine strain can also cause disease in man. It grows more
slowly and less luxuriantly then the human strain.
- some exhibit the presence of Much’s granules
- tubercle bacilli are highy resistant to drying and chemical agents like
acids and alkali because of the hydrophobic nature of the cell wall.
- Virulence factors :
1. Cord factor – glycolipid which can disrupt the mitochondria of a cell
thus decreasing respiration and oxidative phosphorylation of the
affected cell
2. Lipids – responsible for the hydrophobic nature of the organism
3. Mycoloci acid – responsible for the acid fastness of the organism
4. Wax – D – found in the cell wall. It was found to have an adjuvant-
like activity hence is able to retains the organisms in the target
tissues
5. Sulfatides – responsible for the natural red test
- Causative agent of tuberculosis
Tuberculosis may be found in any organ but the usual primary sits of infection are the
lungs. During the first contact with the organism, there will be an acute exudative lesion
which develops rapidly, then spreads to lymphatics and regional lymph nodes. This is
called the primary on the Ghon complex.

When the lesions get stabilized, the bacilli will move to the periphery and the
central area undergoes necrosis. The recrotic area remains semi-solid, like cheese
hence termed as caseous necrosis. When this area liquefies and is expelles by
coughing or sneezing, the tubercle bacilli are aerosolized to form droplet nuclei which
will be the source of infection.
Laboratory identification:
Specimen can be any of the following: sputum
CSF
Urine
Gastric content
Synovial fluid
Pleural fliud
Biopsy materials
1. Stained smears – Look for the presence of AFB (acid-fast bacilli).
Smear may be made directly from specimens or from concentrated
materials.
Concentration of specimens with digesting substances :
a. N-acetyl-L-cysteine with 4% sodium hydroxide (NALC)
b. 4% NaCh
c. 4% H2SO4
d. 20% Chlorox
e. 5.25% sodium hypochlorite
f. Zephiran tri sodium phosphate
g. Oxalic acid
h. Cetyl pyridinium chloride (CPC
Actions of the digesting substaneous : (1) dissolve fat & mucus
(2) kill other organisms other than Mycibacterium
After Acid-Fast staining the smear is studies under the microscope. There are standard
methods of reporting the presence of AFB as to rare. Few or numerous using 800-
1000X magnification (oil immersionlens.)
0 - no AFB
1 – 2/ slide – result is doubtful, repeat smear
3 – 9/ slide – rare
More than 10/ slide – few
More than 1/field – numerous

2. Cultivation – part of the digested material will be be grown in any of the following
media
a. Agar base media : Dube Oliic agar
Middlebreak 7H-10 agar
b. Egg Base Media Petragnani’s medium
Lowenstein-Janssen medium (L-J)
Darset Egg medium
American Trudeau Society (ATS)
Petroff’s medium
Garpen’s medium
In the cultivation observe for the following :
(1) Growth rate – Mycobacterium Tb is a slow grower. It usually require more
than 10 days to develop on solid media. Therefore it is suggested that primary
cultures be inucubated for 6 – 8 weeks before discarding them as negative.
(2) Growth temperature – Mycobacterium Tb grow well at 35 C
(3) Colonial mrorphology – colonies are fry to light buff hones described as
“cauliflower colonies”.
3. Virulence tests :
a. Serpentine cord formation – condensed water from positive culture tube
Mycobacterium tuberculosis when smeared and stained will show the organisms
as bacilli oriented in close parallel arrangements like a serpent.
b. Neutal red dye test – virulent strains of Mycobacterium tuberculosis take up
neutral red (which is yellow in color( in an alkaline medium. Colonies will turn red
after 1 hour incubation in fresh bartibal buffer and the neutral red dye. This
reaction is develop to be due to the presence of strongly acidic lipids and
sulfatides.
c. Niacin production – mycobacterium tuberculosis release large quantities of niacin
into the medium. Presence of niacin is detected with 10% cyanogen bromide in
the color is indicative of niacin.
d. Catalese test – all Mycobacterial isolated produce various quantities of catalese.
Mycobacterium tuberculosis is weakly positive – column foam or bubbles is only
below 45 mm and the catalese produced by the organisms is not stable at 68 C.
4. Tuberculin test – an intradermal test that candetect past or present infection with the
organisms. This test is done with either the :
a. OT – old tuberculin
b. PPD – purified protein derivative

Methods of testing : (1) Mantoux – intracitaneous
(2)Van Pirquet – seratch method
(3) Vollmer – patch method
(4) Moro – percutaneous
(5) Tine – multiple puncture
Prevention : BCG vaccination
Bacillus of Calmette and Guerin I an attenuated mutant of Mycobacterium
bovis used an immunizing agent against tuberculosis.
OTHER Mycobacteria
1. Mycobacterium bovis – produces spontaneous tuberculosis in a wide range of
animals especially the cows. Consumption of milk from the sick animals
has been a common source of infections among human beings. In the
cultivation of M. bovis, glycerol should be omitted from the media
because it selectively inhibits the growth of M. bovis.
2. Mycobacterium ulcerans – produces a destructive primary tropical skin disease
which if not immediately treated will produce chronic ulcers with necrotic
centers.
3. MYCOBACTERIA associated with non-tuberculcus infections
They have been classified by Runyoun into :
1. Slow Growers – growth is observed after 7 days
Group I Photochromogens – colonies are pigmented only after
exposure to light.
Group II Scotochromogens – colonies develop yellow to orange
pigments when grown in the dark but a reddish orange pigment is developed with
continuous exposure to light.
Group III Non-Photochromogens – non pigmented colonies in the
presence of light or in the dark
2. Rapid growers – growth is observed within 7 days

Mycobacterium leprae is the causative agent of lprosy hence it is also known as the
leprosy bacillus which was discovered by Dr. Armauer Hansen so it is also
known as the Hansen bacilli or bacillus of Hanse.
Leprosy is a chronic, specific infection with predilection fr the skin and peripheral
nerves. The earliest symptom is usually an asymptomatic
nypopigmented macule in the trunk or in the extremities. Because
nerves are involved there is usually an anesthetic effect.
Transmission – the current concept accepted by most leprologist is that the
disease is acquired after birth through is that the disease and intimate
contact with a source of infection.
Diagnosis :
1. Demonstration of the acid-fast bacilli in smear of the skins, nasal scripings and
tissues sections.
Lepromatous Leprosy – bacilli are very numerous
Tuberculated leprosy – bacilli are very difficult to detect
2. Lepromin skin test can results in:
a. Fernandez reaction or the early reaction which appears in 24 – 48
hours
b. Mitsuda reaction or the later reaction which appears 3 to 4 weeks after
the injection
Mycobacterium leprae can be distinguished from Mycobacterium tuberculosis in term of
the following :
Mycobacterium leprae M. tuberculosis
1. Smears basilli are packed in mononnclears long and slender arranged
in an arrangement that suggests in chain
a packet of ceigeretters
2. Staining stains more solidly with coarser granules resists ordinary stains
That are widely spaced. It can be Gram
stained
3. Culturability – has never been cultured satisfactory can be cultured in artificial
- has been cultured in mouse foot pads media

Biopsy of skin usually demonstrate the lepra or the Virchow cells, which actually are
historioctes with engulfed masses of leprabacelli. When clumped together, the
lepra cells form a multi nucleate cell called the globi.
AIROBIC SPOREFORMING BACILLI
Bacillus anthrecis - also known as the anthrax bacilli
- large encapsulated non-motile rods
- gram positive : catalese positive
- with square ends hence sometime
- typically form long chains when smears are prepared
from colonies giving the jointed bamboo fishing rod
appearance
- develops an oval spore that tend to be located
centrally producing no detectable swelling of the cell
- can be grown on BAP where colonies are non-
hemolytic, large, rough and opaque with irregular
fringlike edge appearing as long tangled masses of
hair. Others have described them to be comma-
shaped outgrowth hence the terms :
1. medusa head or caput medusa
2. lion head
3. barrister’s wig
- has been successfully cultivated in Polymyxin
medium Ethylandiamine Tetra-acetate medium
(PLET), which is a highly selective medium. Growing
colonies when pushed gently gives a beaten egg
white appearance
- this is the causative agent of Anthrax
anthrax is a disease of animals but from animals the infective agent can be
transmitted to man. There are three-categories of manifestations :
1. cutaneous
2. gentrointestinal
3. pulmonary also known as the Woolsorter’s disease
laboratory identification :
1. Stained smears
2. Cultivation: BAP

PLET
3. Ascali test – used in identifying the organism for animal carcass Tissues
extract will be exposed to anthrax antiserum and at the point of
contact will develop a precipitin bend or ring.
Bacillus Cereus
The organism can produce toxin but if has a limited ability to produce disease. It
has been recognized to be an important cause of food – bore outbreaks of
gastroenteritis. It is uncommonly linked to food poisoning.
Bacillus Anthracis : B. cereus
1. Motility Non-Motile Motile
2. Hemolysis Non-hemolysis B. hemolytic
3. Litmus milk not reduced reduced in 2-3 days
4. Methylene blue not reduced reduced in 24 hours
5. Growth at 45 no growth positive
6. Penicillin sensitivity sensitive resistant
Other Bacillus
1. Bacillus subtilis also known as the Hay bailli. It is another common laboratory
contaminant.
2. Bacillus thuringiensis – used asbiological control of mosquito
3. Bacillus polymyxo – larvae source of antibiotics polymyxin
4. Bacillus licheniformis – source of antibiotics Bacitracin

ANAEROBIC SPOREFORMING BACILLI – Clostridia
The members of this genus may be divided into :
1. Gas Gangrene group composed of :
a. Clostridium perringens also known as Clostrodium welchii
b. Clostridium novvi
c. Clostridium sporogenes
d. Clostridium histolyticum
e. Clostridium bifermentans
f. Clostridium sordelii
2. Toxigenic group :
a. Clostridium tetani
b. Clostridium botulium
General characteristics:
1. All clostridia are motile except Clostridium perfringens.
2. All clostridia are non-encapsulated except C. perfringes.
3. All are singly hemolytic except C. perfringes.
4. All have swollen sporangis except C. perfringes and C. bifermentans.
5. All are lactose non-fermenters except C. perfringes and C. septicum
6. All are sucrose non-fermenters except C. perfringes,
7. All are dextrose (glucose) fermenters except C. tetani and C. histolyticum.
8. Clostridium tetani does not ferment any sugar.
Clostridium tetani also known as the tack head bacilli, drumstick bacilli, lollipop bacilli
and the tennis racket bacilli because of the terminal spore whose diameter is wider than
the organism.
- it is the causative agent of tetanus
tetanus is also known as lockjaw or trismus, classic symptom of which is muscle
stiffness of the lower jaw followed by spamsm of the masseter muscles.
Tetanus occurs because of the toxins elaborated by the organism namely :
1. tetanolysin – a hemolysin
2. tetanospasmin – a neutrotoxin responsible for the stiffness and spasms of
musles involved.
1. Stained smears from cultures shows the typical spherical terminal spore.

2. Anaerobic cultivation
On BAP, the organisms produce beta type of hemolysis
Prevention – Giving DPT vaccination
D for diphtheria caused by Corynebacterium diphtheria
P for pertussi caused by Hemophilus pertussi
T for tetanus cause Clostridium tetani

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