Download Microbiologic Examination

Microbiologic Examination
 Direct Examination
 Direct examination of specimens frequently
provides the most rapid indication of microbial
infection.
 A variety of microscopic,
immunologic, and
hybridization techniques have been developed for
rapid diagnosis
Staining
 Staining is a technique used in microscopic
techniques used to enhance the clarity of
the microscopic image. Stains and dyes are
widely used in the scientific field to highlight
the structure of the biological specimens,
cells, tissues etc.
Gram stain
 The most widely used staining procedure in microbiology is
the Gram stain, discovered by the Danish scientist and
physician Hans Christian Joachim Gram in 1884. Gram
staining is a differential staining technique that differentiates
bacteria into two groups:
Gram-positives and gram-negatives. The procedure is
based on the ability of microorganisms to
retain color of the stains used during the gram
stain reaction.
 Gram-negative bacteria are decolorized by the alcohol, losing the color
of the primary stain, purple.
 Gram-positive bacteria are not decolorized by alcohol and will remain
as purple. After decolorization step, a counterstain is used to impart a
pink color to the decolorized gram-negative organisms.
Gram staining
 Objectives:
 To
differentiate between the two major
categories of bacteria: Gram positive and Gram
negative.
 To understand how the Gram stain reaction
affects Gram positive and Gram negative bacteria
based on the biochemical and structural
differences of their cell walls.
Gram staining cont……
 Gram positive bacteria: Stain dark purple
due to retaining the primary dye called
Crystal Violet in the cell wall.
Example: Staphylococcus aureus
 Gram negative bacteria: Stain red or pink
due to retaining the staining dye called
Safranin.
Example: Escherichia coli
Gram Stain Mechanism
 Gram Positive Cell Wall:
 Gram-positive bacteria have a thick mesh-like cell wall
which is made up of peptidoglycan (50-90% of cell
wall), which stains purple. Peptidoglycan is mainly a
polysaccharide composed of two subunits called Nacetyl glucosamine and N-acetyl muramic acid. As
adjacent layers of peptidoglycan are formed, they are
cross linked by short chains of peptides by means of a
transpeptidase enzyme, resulting in the shape and
rigidity of the cell wall. The thick peptidoglycan layer
of Gram-positive organisms allows these organisms to
retain the crystal violet-iodine complex and stains the
cells as purple.
Gram Stain Mechanism
Gram Negative Cell Wall:
 Gram-negative bacteria have a thinner layer of
peptidoglycan (10% of the cell wall) and lose the
crystal violet-iodine complex during decolorization
with the alcohol rinse, but retain the counter stain
Safranin, thus appearing reddish or pink.
The four basic steps of the
Gram Stain
 1) Application of the primary stain Crystal
Violet (CV) to a heat-fixed smear of
bacterial culture.
 CV dissociates in aqueous solutions into CV+
and Cl – ions. These two ions then penetrate
through the cell wall and cell membrane of
both Gram-positive and Gram-negative cells.
The CV+ ions later interacts with negatively
charged bacterial components and stains the
bacterial cells purple.
The four basic steps of the
Gram Stain cont….
 2) Addition of Gram’s Iodine.

Iodine acts as a mordant and as a trapping
agent. A mordant is a substance that increases
the affinity of the cell wall for a stain by binding
to the primary stain, thus forming an insoluble
complex which gets trapped in the cell wall. In
the Gram stain reaction, the crystal violet and
iodine form an insoluble complex (CV-I) which
serves to turn the smear a dark purple color. At
this stage, all cells will turn purple.
The four basic steps of the
Gram Stain cont….
 3) Decolorization with 95% ethyl alcohol.(Gram
negative bacteria)
 Alcohol or acetone
 dissolves the lipid outer membrane of Gram negative
bacteria, thus
 leaving the peptidoglycan layer exposed and
 increases the porosity of the cell wall.
 The CV-I complex is then washed away from the thin
peptidoglycan layer,
leaving Gram negative bacteria
colorless.
The four basic steps of the
Gram Stain cont ……
 3) Decolorization with 95% ethyl alcohol .(Gram
positive bacteria )
 On the other hand, alcohol has a dehydrating effect on the cell
walls of Gram positive bacteria which causes the pores of the cell
wall to shrink. The CV-I complex gets tightly bound into the
multi-layered, highly cross-linked Gram positive cell wall thus
staining the cells purple.
 The decolorization step must be performed carefully, otherwise over-
decolorization may occur. This step is critical and must be timed
correctly otherwise the crystal violet stain will be removed from the Grampositive cells. If the decolorizing agent is applied on the cell for too long
time , the Gram-positive organisms to appear Gram-negative.
 Under-decolorization occurs when the alcohol is not left on long
enough to wash out the CV-I complex from the Gram-negative cells,
resulting in Gram-negative bacteria to appear Gram-positive.
Decolorizing agents
 Various formulations of decolorizing agents
may be used (acetone, acetone/ethanol,
ethanol).
 Acetone is the most rapid decolorizer
 Ethanol is recommended for student use to
prevent over-decolorization of samples
The four basic steps of the
Gram Stain cont……
 4) Counterstain with Safranin
 The decolorized Gram negative cells can be
visible with a suitable counterstain, which is
usually positively charged safranin, which
stains them pink.
 Pink colour which adheres to the Gram positive
bacteria is masked by the purple of the crystal
violet
Gram Stain PROTOCOL
1. air-dried, heat-fixed smear of cells
2.
Flood slide with for 1 minute with crystal violet staining reagent.
Please note that the quality of the smear (too heavy or too light cell
concentration) will affect the Gram Stain results.
3.
4.
Wash slide in a gentle and indirect stream of tap water for 2 seconds.
Flood slide with the mordant: Gram's iodine. Wait 1 minute.
5.
Wash slide in a gentle and indirect stream of tap water for 2 seconds.
6. Flood slide with decolorizing agent (ETHANOL). Wait 15 seconds or
add drop by drop to slide until decolorizing agent running from the
slide runs clear .
7. Flood slide with counterstain, safranin. Wait 30 seconds to 1 minute.
8. Wash slide in a gentile and indirect stream of tap water until no color
appears in the effluent and then blot dry with absorbent paper.
Observations
 Gram-stained bacteria should be viewed with
a brightfield microscope at 1000X
magnification with oil immersion. If the
smear of cells is crowded it will be difficult to
note cell shape and arrangement.
Techniques
 For microscopic examination it is sufficient
to have a compound microscope equipped with
 Low-power (1OX),
 High-power (40X), and
 Oil immersion (1OO0X)
objectives,
 A mechanical stage,
 A substage condenser, and
 A good light source.
 For nucleic acid study
 For immunological studies
achromatic
Techniques cont…..
 Genetic probes are based on the detection of
unique nucleotide sequences with the DNA or RNA
of a microorganism.
 Once such a unique nucleotide sequence, which
may represent a portion of a virulence gene or of
chromosomal DNA, is found, it is isolated and
inserted into a cloning vector (plasmid), which is
then transformed into Escherichia coli to produce
multiple copies of the probe. The sequence is then
reisolated from plasmids and labeled with an
isotope or substrate for diagnostic use.
Techniques cont…..
molecular technology in the
diagnoses of infectious diseases has been further
 The use of
enhanced by the introduction of gene amplification
techniques, such as the polymerase chain reaction (PCR)
in which DNA polymerase is able to copy a strand of DNA
by elongating complementary strands of DNA that have
been initiated from a pair of closely spaced
oligonucleotide primers.
major applications in the
detection of infections due to microorganisms
 This approach has had
that are difficult to culture (e.g. the human immunodeficiency
virus) or that have not as yet been successfully cultured
Culture
 In many instances, the cause of an infection is
confirmed
by
isolating
and
culturing
microorganism either in artificial media or in a
living host.
 Bacteria
(including
mycobacteria
and
mycoplasmas) and fungi are cultured in
either liquid (broth) or on solid (agar)
artificial media.
 Liquid media provide greater sensitivity
for the isolation of small numbers of
microorganisms
 Identification
cultures
of
mixed
growing in liquid media
requires subculture onto solid media so
that isolated colonies can be processed
separately for identification.
 Growth in liquid media also cannot
ordinarily be quantitated. Solid
media, provide isolated colonies that
can be quantified if necessary and
identified. Some genera and species
can be recognized on the basis of
their colony morphologies.
Identification of mixed cultures Cont……
 In some instances one can take advantage of
differential
fermentation
carbohydrate
capabilities of microorganisms by
incorporating one or more carbohydrates in the
medium along with a suitable pH indicator. Such media
are called
differential media (e.g., eosin
methylene blue or MacConkey agar)
and are commonly used to isolate enteric bacilli.
Different genera of the Enterobacteriaceae
can then be presumptively identified by the
color as well as the morphology of colonies.
Identification of mixed cultures cont…..
 Culture media can also be made selective by incorporating
compounds such as antimicrobial agents that inhibit the
indigenous flora while permitting growth of specific
microorganisms resistant to these inhibitors.
 One such example is Thayer-Martin
medium, which
is used to isolate Neisseria gonorrhoeae and N meningitidis.
This medium contains :
 vancomycin to inhibit Gram-positive bacteria,
 colistin to inhibit most Gram-negative bacilli,
 trimethoprim-sulfamethoxazole
to
inhibit Proteus species and other species that
are not inhibited by colistin
 anisomycin to inhibit fungi.
The pathogenic Neisseria species, N gonorrhoeae and N meningitidis,
are ordinarily resistant to the concentrations of these antimicrobial
agents in the medium.
 The
number of bacteria in
specimens may be used to define
the presence of infection
 Chlamydiae and viruses are cultured in
cell culture systems
 virus
isolation
occasionally requires
inoculation into animals, such as suckling mice,
rabbits, guinea pigs, hamsters, or primates.
 Some
viruses,
viruses,
such
as
the
hepatitis
cannot be isolated in cell culture
systems, so that diagnosis of hepatitis
virus infection is based on the
detection of hepatitis virus antigens or
antibodies.
 Cultures are generally incubated at 35 to 37°C in




an atmosphere consisting of air,
air supplemented with carbon dioxide (3 to 10 percent),
reduced oxygen (micro aerophilic conditions), or
no oxygen (anaerobic conditions), depending upon
requirements of the microorganism.
 clinical specimens from bacterial infections often
contain:
 aerobic,
 facultative anaerobic,
 and anaerobic bacteria,
such specimens are usually inoculated into a variety of
general purpose, differential, and selective media, which
are then incubated under aerobic and anaerobic
conditions
General procedure for collecting and
processing specimens for aerobic and/or
anaerobic bacterial culture.
Medical Microbiology. 4th
edition.
Baron S, editor.