Introduction to diagnostic bacteriology
Introduction
The primary mission of the clinical bacteriology laboratory is to assist the
health care provider in the diagnosis of infectious diseases.
Clinical bacteriology focuses on all aspects of patient infections;
a) from testing samples to identifying bacterial agents that are
contributing to the infectious process,
b) to determining the susceptibility of microorganisms to various
antimicrobial agents, and
c) reporting the results to the physician.
• The job of the clinical microbiology laboratory include;
1. To test specimens from patients for microorganisms that are, or
may be, a cause of the illness.
2. To provide information (when appropriate) about the in vitro
activity of antimicrobial drugs against the microorganisms
identified.
3. confirming a clinical diagnosis of infectious disease with a
bacterial etiology.
4. The clinical microbiologist should participate in decisions
regarding the microbiological diagnostic studies to be performed.
5. Advise on the type and timing of specimens to be collected.
6. Mode of transportation and storage.
7. Above all, the clinical microbiology laboratory, whenever
appropriate, should provide an interpretation of laboratory results.
Laboratory procedures used in the diagnosis of infectious diseases
in humans include the following:
-Morphologic identification of the agent in stains specimens or
tissues using light and electron microscopy.
-Culture isolation and identification of the agent.
-Detection of antigen from the agent by immunologic assay (
Latex agglutination, EIA, ELISA etc).
-PCR method (DNA/DNA, DNA/RNA hybridization).
-Antibody demonstration.
Specimen Selection, Collection and Processing
A properly collected specimen is the single most important step in
the diagnosis of any disease.
This is because the results of diagnostic tests for infectious
diseases depend upon selection, timing, and method of collection
of specimens.
The specimen must be collected from the anatomical site most
likely to yield the agent at that particular stage of illness.
Besides, the specimen must be handled in such a way that will
favour the organisms survival and growth.
General rules to all specimens:
1. The quantity specimen must be adequate.
2. It should be representative of infectious process e.g.,
sputum not saliva, swab from wound’s depth not from surface.
3. Avoiding specimen contamination by using only sterile
containers and observing aseptic precautions.
4. Prompt transportation of specimen to the laboratory and
examination must be immediately.
5. Samples must be taken or collected before administering
drugs.
6. Specimens obtained through operation require special
attention ; Enough tissue must be obtained for both
histopathologic and microbiologic examination.
Histopathologic examination is used to distinguish
neoplastic from inflammatory lesions and acute from chronic
inflammations.
The type of inflammation present can guide the type of
microbiologic examination performed.
If, for example, a caseous granuloma is observed
histopathologically, microbiologic examination should
include cultures for mycobacteria and fungi.
6. Ovoid normal bacterial flora during sample collection
especially the mouth, nose, skin and other surfaces.
• This can be a nuisance in that ;
✓ It can contaminate specimens
✓ It may cause infection (Opportunistic infection) , if they
access to deep tissues by trauma , surgery, lines or in
immunocompromised patients , e.g., S epidermidis,
Acinetobacter etc
.
Getting the specimen to the Lab
• Problem in delay or inappropriate storage.
• Delay in diagnosis & treatment lead to;
✓ Pathogens die
✓ Contaminant's overgrowth
• Blood culture directly into incubator not refrigerator
• CSF straight to lab
• Don’t put the entire surgical specimen in to formalin; but send
a portion to microbiology in a sterile container
Specimen processing
❖ Receiving
❖ Recording
❖ Culturing
❖ Staining
❖ Isolation
❖ Identification
❖ Susceptibility testing
Receiving & Recording
• Patient details (Name, age and gender)
• Laboratory number/Hospital Number/Unique identification number
• Location (OPD/Ward etc)
• Type of specimen
• Collection date and time
• Test requested
• Clinical history (Suspected diagnosis)
Receiving & Recording
• Travel history
• Immunization if any
• Be considerate to lab staff !!
✓ Label hazardous specimen
• Don’t send specimen to the lab without proper packing
✓ Leaking or blood-stained specimens are not acceptable !!
Receiving & Recording
• Travel history
• Immunization if any
• Be considerate to lab staff !!
✓ Label hazardous specimen
• Don’t send specimen to the lab without proper packing
✓ Leaking or blood-stained specimens are not acceptable !!
Factors limiting usefulness of bacteriological
investigations
• Wrong sample e.g., saliva instead of proper sputum
• Delay in transport / inappropriate storage e.g., CSF
• Overgrowth by contaminants e.g., Urine
• Insufficient sample or sampling errors
• Collection of sample, while patient has received antibiotic
Specimen rejection criteria
• Mismatched information
• Improper container or temperature
• Insufficient specimen
• Leaking specimen
• Formalin specimen
• Dry swab
• Late specimen
✓ Physician must be informed about the rejection
Methods of identification
There are different methods used in the diagnosis of diseases
i.e.
1. Macroscopy
2. Microscopy
3. Culture
4. Sensitivity tests
5. Serological tests
6. Molecular detection
Macroscopic Examination
• By naked eyes observation such as;
Turbidity
Hemolysis
Physical appearance (eg in sputum saliva vs mucopurulent)
Colony morphology (In culture plate) etc
Microscopic Examination
• Prepare and examine slides by the direct wet film and iodine-
stained procedures.
•STAINING METHOD
Microbiologic Examination and stains
Microscopic examination of both stained and unstained
specimens is simple and inexpensive but less sensitive when
compare with culture for detection of small numbers of bacteria.
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.
Routine staining for Microscopic Examination
➢ Sample stain
➢ Gram stain
➢ Acid fast stain ( Ziehl Nilsen stain)
➢ Special stains (Gimesa etc)
➢ Immunofluorescence techniques
Culturing of Bacteria
A microbiological culture, or microbial culture, is a method of
multiplying microbial organisms by letting them reproduce in
predetermined culture medium under controlled laboratory conditions.
Microbial cultures are foundational and basic diagnostic methods used
in diagnostic bacteriology.
In many instances, the cause of an infection is confirmed by isolating
and culturing microorganism either in artificial media or in a living host.
Some genera and species can be recognized based on their colony
morphologies.
Antimicrobial Susceptibility
The responsibility of the microbiology laboratory includes not
only microbial detection and isolation but also the
determination of microbial susceptibility to antimicrobial
agents.
Many bacteria have unpredictable susceptibilities to
antimicrobial agents, and their susceptibilities can be
measured in vitro to help guide the selection of the most
appropriate antimicrobial agent.
Antimicrobial susceptibility tests are performed by
1. Disk Diffusion Method
2. Minimum Inhibitory Concentration (MIC)
3. E test
Bacteria Susceptible to antibiotic
The term susceptible means that the microorganism is inhibited by a
concentration of antimicrobial agent that can be attained in blood
with the normally recommended dose of the antimicrobial agent and
implies that an infection caused by this microorganism may be
appropriately treated with the antimicrobial agent.
Bacteria resistant to antibiotic
The term resistant indicates that the microorganism is resistant
to concentrations of the antimicrobial agent that can be attained
with normal doses and implies that an infection caused by this
microorganism could not be successfully treated with this
antimicrobial agent.
Immunological assay
The most frequently used techniques for immunologic
detection of microbial antigens in the laboratory include, latex
particle agglutination and enzyme-linked immunosorbent
assay (ELISA).
Molecular techniques
The use of molecular technology in the diagnoses of infectious
diseases has been further enhanced by the introduction of gene
amplication techniques, such as the polymerase chain reaction
(PCR).
In PCR DNA polymerase can copy a strand of DNA by elongating
complementary strands of DNA that have been initiated from a pair
of closely spaced oligonucleotide primers.
This approach has had major applications in the detection of
infections due to microorganisms that are difficult to culture (e.g.,
the human immunodeficiency virus) or that have not yet been
successfully cultured .
Summary
Information provided by microscopic techniques serves as a
rapid presumptive diagnosis of an infection e.g., pulmonary
tuberculosis using ZN staining techniques, gram staining
technique is used to distinguish whether a micro-organism
(bacteria) is either gram positive or gram negative, a coccus or
bacillus.
Useful information's can also be provided from the microscopic
examination of wet preparations e.g., when looking for motile
vibrios in a fecal specimen or capsulated C neoformans in CSF.
Other diagnostic techniques i.e., culture isolation and
identification of the agent, detection of antigen from the agent by
immunologic assay ( Latex agglutination, EIA, ELISA etc), PCR
method (DNA/DNA, DNA/RNA hybridization) and antibody
demonstration (serology) are equally play important role
depends on the availability.
