Laboratory Methods For Identification Of Bacteria
Bacteria are either identified in:
1. A pathological specimen obtained from the patient (e.g. pus, sputum,
urine, blood, stools, etc.) depending on the site of infection.
2. After been grown on artificial nutrient media.
Bacteria are then identified by:
(I) Microscopic Examination:
a.
Examination of fresh samples used for demonstration of bacterial
motility; using hanging drop method.
b.
Morphology and staining reactions of bacteria.
Commonly used stains are:
1. Simple stains: e.g. methylene blue stain.
2. Differential stains: e.g.
i. Gram stain:
1) Primary stain: Methyl violet- Iodine mixture.
2) Decolourization with Alcohol.
3) Counter stain: Diluted carbol fuchin.
Results:

Gram (+) Purple
 Gram (-)
Red
 Difference - due to structure of cell wall
• Gram (+) Thick cell wall
• Gram (-)
Thin cell wall
a
ii. Acid fast stain: (Ziehl-Neelsen stain)
Differential Stain - divides bacteria into 2 groups
 Acid - Fast
 Non Acid - Fast
Used to identify organisms in the Genera Mycobacterium (high lipid
and wax content in cell wall)
1. Carbol fuchsin (Red)
2. Decolourization by sulphuric acid 20%.
3. Counter stain with Methylene blue.
Acid - Fast organism: Red as Mycobacterium tuberculosis
Non Acid – Fast organism: Blue
iii. Special stains:
e.g. Capsule stain, flagella stain, …..
(II) Cultural Characters:
Bacteria need nutritive culture media to multiply in vitro.
An undefined medium (also known as a basal or complex medium). It
is a medium that contains:
1- A carbon source such as glucose for bacterial growth
2- Water
3- Various salts needed for bacterial growth.
Defined media (also known as chemically defined media or synthetic
media).
Classification of Media:
Media can be classified into:
1-Minimal media (simple medium):
It contains the basic nutritive requirements, e.g. nutrient broths and
agar media.
2- Selective media:
Selective media are used for the growth of only selective microbes.
It contains antibiotics, dye, or specific chemicals that inhibits the
growth of most types of microbe and stimulate the isolation of one
type.
 Mannitol salt agar (MSA) which is selective for Gram +_ve
bacteria.
 Blood-free, charcoal-based selective medium agar (CSM) for
isolation of Campylobacter.
 Lowenstein- Jensen medium:
enriched selective media for T.B.
 T.C.B.S (Thiosulphate- CitrateBile- Sucrose agar): selctive for
Vibrio cholera due to alk. pH.
3-Differential media:
Differential media or indicator media distinguish one microorganism
type from another growing on the same media.
Indicators (such as neutral red, phenol red, eosin y, or methylene blue)
Examples of differential media include:
Eosin methylene blue (EMB), which is differential for lactose and
sucrose fermentation.
MacConkey (MCK), which is differential for lactose fermentation.
4- Enriched media:
Enriched media contain the nutrients required to support the growth
of a wide variety of organisms, including some of the more
fastidious ones.
Blood agar : Is an enriched medium in which nutritionally rich
whole blood supplements the basic nutrients. It contains 5-10%
human or animal blood.
It shows the type of haemolytic activity of bacteria (complete, partial
or non- haemolytic).
Complete
Partial
Haemolysis of
Haemolysis
RBCs
of RBCs
(Beta
(Alpha
Haemolytic
Haemolytic
Streptococci)
Streptococci)
Chocolate agar (heated blood agar):
is enriched with heat-treated blood (40-45°C).
Lofflers serum media:
Horse serum + glucose in a ratio 3:1
It is used for cultivation of Corynbacterium diphtheria.
5- Transport media:
Transport medium is a simple organic medium that Maintain the
viability of all organisms in the specimen without altering their
concentration.
This type of medium mainly used for Temporary storage of
specimens being transported to the laboratory for cultivation.
Examples of transport media include:
Thioglycolate broth for strict anaerobes.
The colonial appearance on culture media:
Shape:
The colonies may be small (pin-point) fimbriate, flat or convex.
Colour:
• The colonies may be colourless or bacteria produce endopigments
which give the colonies a characterestic colour , e.g.
Staph. aureus produce golden yellow colonies.
Staph. albus produce white endopigment.
Staph. Citreus produce a lemon yellow endopigment.
• The bacteria may produce exopigments,e.g. Pseudomonas
aeruginosa produce a green exopigment in the surrounding media.
Antimicrobial Chemotherapy:
An antibacterial agent is a compound or substance that kills or slows
down the growth of bacteria.
Antibiotic(s) has come to include a broader range
of antimicrobial compounds, including anti-fungal and other
compounds.
It is produced by microbes and is harmful to other microbes, except
viruses.
These include, for example, the beta-lactam antibacterials, which
include the penicillins (produced by fungi in the genus Penicillium
notatum),and the cephalosporins.
Compounds that are still isolated from living organisms are the
aminoglycosides, whereas other chemotherapeutic agents—for
example, the sulfonamides,and the quinolones, are produced by
chemical synthesis.
Classification of Antibiotics:
According to agent action:
In this classification antibacterial agents are divided into two broad
groups according to their biological effect on
microorganisms: bactericidal agents kill bacteria, and bacteriostatic
agents slow down or stall bacterial growth.
Bactericidal antibiotics:
Antibiotics that inhibit cell wall synthesis: the Beta-lactam antibiotics
(penicillin derivatives, and cephalosporins).
Aminoglycosidic antibiotics are usually considered bactericidal,
although they may be bacteriostatic with some organisms.
Bacteriostatic antibiotics limit the growth of bacteria by interfering
with bacterial protein production, DNA replication, or other aspects
of bacterial cellular metabolism.
This group includes:
Tetracyclines, sulphonamides , trimethoprim ,chloramphenicol,
and macrolides.
Antibiotic sensitivity test:
Antibiotic sensitivity is a term used to describe the susceptibility
of bacteria to antibiotics.
Antibiotic susceptibility testing (AST) is usually carried out to
determine which antibiotic will be most successful in treating a
bacterial infection in vivo.
Testing for antibiotic sensitivity is often done by the Kirby-Bauer
method ( Disc-diffusion method).
Other methods to test antimicrobial susceptibility include the Etest (also based on antibiotic diffusion).
Agar and Broth dilution methods for Minimum Inhibitory
Concentration determination.
Antibiotic sensitivity test:
Antibiotic sensitivity Test : Different antibiotics have been placed
on an agar plate growing bacteria and the plate is incubated. The
degree of inhibition of growth around the discs is measured.
The more the zone of inhibition, the more the sensitivity to the
antibiotics
The Dilution Method:
Serial dilutions of antibiotics are incorporated in agar containing or
broth culture media.
The lowest concentration of antibiotic that prevents visible growth
after an 18-24 hours incubation period is known as minimal
inhibitory concentration (MIC).
The minimal bactericidal concentration (MBC) may be determined in
broth dilution tests by subculturing the containers that show no
growth on to antibiotic-free agar containing media.
The lowest concentration of antibiotic that totally suppresses growth
after overnight incubation is known as MBC.