MCB 602 LECTURES

• MCB 602 COURSE OUTLINE

• COURSE TITLE: CLINICAL MICROBIOLOGY

• Instructors: Roland Ndip /Lucy Ndip / Irene
Anyangwe/ Seraphine Esemu
Objectives: To orient the students on the
collection, handling, transportation and culture of
specimens, serodiagnostic procedures and
antimicrobial chemotherapy (This aspect is
extensively covered in MCB 606). important
viruses, fungi and rare bacterial infections.
•
• It is logical to exclude this and focus on other
areas not covered in the general programme. This
is addressed from weeks 6-8 to include clinically
important viruses, fungi and rare bacterial
infections.

• Content: Method of collection, handling,

transportation and culture of various clinical
specimens (blood, stool, urine, CSF etc);
Serodiagnostic techniques; nosocomial infections;
antimicrobial chemotherapy; susceptibility
testing; various groups of antimicrobial agents
and their modes of action.
• Outcome: Knowledge on collection, handling
and analysis of clinical specimens as well as
diagnostic techniques and the management of
infections

Lecture 1
• Introduction:
-General issues on Clinical Microbiology
Laboratory
-Isolation and identification of medically
important organisms
• Lecture 2
• Collection and transportation of laboratory specimens
• -Throat and Nasopharyngeal specimens
• -Sputum
• -Ear and Eye Specimen
• -Stool and rectal swab
• -Genital tract specimen
• - Skin lesions
• -Wound
• - Biopsy material
• -Cerebrospinal fluid
• -Blood
•
• Lecture 3
• Laboratory examination of clinical specimens
• - Microscopy
• - Culture
• - Biotyping
• - Serotyping
• -Antibiogram
• -Molecular typing
• Lecture 4
• Nosocomial infections
• - Definition
• -Predisposing factors
• - Pathogens and site of infection
• -Routes of transmission
• -Prevention and control

• Lecture 5
• -Test
• Lecture 6: Clinically important Viruses
• -Adenovirus
• - Human Papilloma Virus
• -Lentiviruses
• -Herpes/Alpha viruses
• -Rotavirus
• -Enteroviruses
• -Hepatitis viruses
• Filoviruses
• Lecture 7: Clinical mycology
• -Opportunistic fungi
• -Systemic fungi
• -Dermatophytes

• Lecture 8: Rare bacterial agents of clinical

importance
• - Chlamydia
• - Rickettsia
• - Mycoplasma
• Lecture 9:
• - Revision

• Lecture 10:
• -Practicals
Laboratory Management
• -Human resource management
• Managers need to understand human behaviour
and manage people and incorporate the
following into their managerial skills
-Motivation
-Perception
-Communication
-Leadership/management skills
-Group dynamics
-Morale
• Planning and Implementation
• Management must plan to make things work
taking the following into consideration:
-The business plan: Lab missions, goals and
objectives
-The marketing plan: customers, test products,
services
-Operation plan: facilities, equipment, capacity
-Staffing plan: workload, staffing requirement
-Financial plan: capital, financial resources, budget
etc.
It is important to note that cost is a critical element
of the Lab’s planning process.
• Control
• After the plan is implemented, management must
monitor or evaluate the overall process and product
quality, compare the results to the initial goals and
expectations, and explore possible alternatives.
• Improvement
• Quality improvement is a management tool used to:
-define customer’s expectations
-describe and evaluate the processes used to provide
service
-continuously improve these processes and outcomes
• QI should focus on the customer’s need rather
than process problems and should rely on
training and prevention to improve service.

• Determination and Management of Lab Cost

• Budget
--This is the primary document that summarizes
the fiscal management of the Lab
-The budget can be a deceptive document in that
it can create rigid guidelines for the activities of
the Lab, or can be used generally with
expectation that only predetermined goals will
be produced.
• Cost Accounting
• This makes it possible to respond to budgetary
needs.
• There are options available to a Lab director
-Traditional cost-accounting system: considers
global costs incurred per section and determine
cost per test.
-Activity –based cost accounting: more focused
and determines the cost involved with each
activity of the Lab. This method is however very
expensive to run. Amount of data which must be
collected, stored, and manipulated in order to
inform decision may be demanding.
• Laboratory Cost
• Could be direct and indirect
• Direct
-This could be arbitrary but should include the
cost of labour including benefits, supplies, and
reagents, as well as, allocation for instrument
depreciation, maintenance, rental, or lease cost
• Indirect Cost
-This will include all other expenses necessary to
do business and are not directly related to a
specific instrument or test.
• Analysis of Laboratory Activities
• Management Techniques
• The primary goal of the CML is to provide
accurate diagnostic testing and high-quality
service at the lowest cost to its customers.
• Achieving this requires a detailed analysis of the
Lab processes and products.
• Management must define the Labs goals, which
may include:
-reduction of reagent and labour costs
-reduction of lengths of stay for patients
-improvement of productivity
 -improvement of TATs for tests
-improvement of the quality of specimens submitted
-improvement of the clinical relevance of test results.
• After the Labs goals have been defined, the methods
to change the Labs operation must be decided. There
are four broad approaches :
-reengineering: critical rethinking and redesign of
systems to produce, deliver and support patient care
-downsizing or reorganizing: reducing cost by laying off
employees
-process improvement: continuous QI.
-systems analysis: improving productivity and efficiency
• By reducing costs, usually based on these:
-equipment and technology
-human resources
-reagents and supplies
-space.
• Areas of operations in CML that may provide
opportunities for some cost reduction include
-Automation: use of machines to redistribute
workload; low numbers of staff or low-skilled
-Alternative technologies or methods: replace
traditional multistep methods by more rapid and
accurate tests that reduce overall costs
• Specimen acceptability: accepting only
specimens that are properly collected,
transported, and labelled and that are
appropriate for the Lab diagnosis of an
infectious disease.
• Extent of specimen workup and organism
identification: complete workup of specimens
had contributed to our understanding of
disease process, provided evidence for
nosocomial spread and the discovery of new
pathogens and emerging antimicrobial
resistance.
-Severity of the disease
-type of specimen submitted
-etiologic agents sought
-clinical management of patient
 Laboratory Design
• A well design lab is a safe, pleasant, and efficient place
in which to work, as well as an enjoyable place to visit.
• When designing a renovating a lab., the needs must:
- meet the needs of the staff who spend all or most of
their working time there;
- workers who spend part of their working time there
- visitors who have to interact with the staff.
• A pleasant environment improves staff morale and
productivity, minimizes unnecessary distraction,
thereby reducing errors, and enhances employee
recruitment and retention.
• An efficient CML helps to:
-improve productivity
-reduce errors
-improves patient care by shortening lab test
turnaround time (TAT)
-improves staff morale and contributes
significantly to pleasant work environment.
• When designing a CML, it should be recalled
that there are three key differences between
a CML and other types of clinical labs.
• First, clinical Microbiol involves the isolation,
propagation, and handling of pathogenic orgs
that pose a risk to lab personnel; hence CML
should have facilities and processes necessary
to meet BSL 2 and to an extent BSL 3.
• Second, the interpretation of cultures, and
other microbiologic test results, is based on
the ability of the lab to isolate pathogenic orgs
while minimizing contamination.
• Third, lab design must accommodate
specialized equipment used only in
microbiology labs.
• General Design Principles
• The needs of each lab will change over time
between the initial construction of the lab and
subsequent renovations.
• The best way to is follow a generic design that
is easy to meet.
• Laboratory Location:
-CML s providing services for a hospital must
be fully integrated with the main hospital lab.
This is critical in providing adequate staffing,
ensuring proper specimen processing and
• Although off-site labs also have their place in
serving the public, they are limited in that:
- there may be delays in specimen processing
which may affect microbial recovery and
timely result reporting.
-decrease in interaction between
microbiologist and clinicians , which may also
affect processing and results.
-loss of integration with the rest of the clinical
lab, particularly specimen receipt and
processing areas, increases the risk of
incorrect specimen processing and medical
-Lab staff members in different lab sections are
unable to interact on a frequent basis, provide
coverage for one another, and act as a
cohesive unit.
- added complexity and cost associated with
reliable and timely transport of specimens.
-location of the CML close to the main lab
facilitates access during off-hours
- location of the CML distant to patient care
precludes meaningful training of clinical
microbiologists, infectious disease physicians,
• Laboratory Size
• As a general rule , each bench technologist
needs about 5 sq M in which to work,
excluding space for large pieces of equipment,
walls, corridors, storage, lockers , and offices.
• Areas such as that for specimen processing
require adequate space to take equipment,
specimen receiving bench and foot traffic.
• Lab sections such as mycobacteriology,
mycology, and virology require a larger a
larger amount of space relative to the
• Labs should be designed with more space
than is needed for current workloads and
types of services provided to allow for
expansion in future.
• Transportation
-As with other lab sections, an efficient
transportation system is crucial for providing
needed services.
-Systems in use include manual, robotic and
pneumatic tube systems, with their
advantages and disadvantages.
• Specific Design Issues
• Laboratory Layout
• Lab layout is crucial for achieving the goals of Lab.
• The overall layout is determined by:
-types of services provided
-numbers and types of specimens processed
-physical constraints of the building
-resources available for construction and renovation
• A Lab that provides only patient care has needs
different from those of teaching/research
• Busy clinical Lab must be efficient to maximize
specimen throughput and minimize TAT
• Labs that support teaching/research missions
also need to be efficient but must have the
space and facilities to support the broader
missions.
• Most CMLs are divided into sections
according to the way that specimens are
handled, eg,
-bench devoted to urine cultures
-bench devoted to blood cultures
• Specimen Preparation Area
• All CMLs need an area to receive and process
specimens.
• This area should be located near the lab entrance so
that other lab staff and courier do not need to enter
the lab to drop off specimens
• The area should have ample benchtop space for
receiving specimens, for any necessary equipment,
and for the handling of specimen requisitions.
• It should have a class II biological safety cabinet in
which all initial specimen processing should be done.
• All necessary IT infrastructure and
telecommunications equipment should be
provided.
• It should have a sink for hand washing and for
performing Gram’s stain and other direct
exams.
• Microscopes could also be placed here for
reading slides.
• A refrigerator should be located here to hold
specimens and media.
• Important to note that the design of this
• General Lab Bench Space
• Work Areas
• Most labs are constructed on the basis of U-Shaped modules
or linear benches.
• Modules usually measure 3 by 3M and can accommodate
two to three persons.
• Advantages to the modular approach include:
-minimized foot traffic in work areas
-generous countertop and storage space
-corner space available for computer workstations
-an increased sense of privacy for workers
-use of less floor space for aisles and corridors
• Advantages to the linear bench approach
include:
• -ease of cleaning
• -ease of moving about the lab
• - easier location of large pieces of equipment
as such incubators and refrigerators
• -ease of moving modular casework, and lower
design, construction, and renovation costs.
• Some labs however use a combination of
modules and linear benches .
• In many cases, the physical layout and
• To accommodate ample waste containers both
for paper and hazardous waste.
• The lab should contain space for storing
completed cultures , st0ck cultures, reference
books, and teaching materials.
• Important to note that it is efficient for
completed cultures and reference materials to
be located close to workbenches, while stock
cultures should be in a secure location ,
especially if the lab maintains reference stocks
of potential biothreat agents such Brucella
spp, Bacillus anthracis, Francisella tularensis,
• Air, Gas, and Vacuum Supplies
• Most modern labs have little need for
compressed air, gas, and vacuum supplies,
except as needed for specific purposes.
• Use of open flames should be prohibited, so
there is no need for a flammable gas supply.
• Central systems for the supply of gases used in
microbiological incubators should be included
within the lab facility.
• A generous amount of space should be
allocated for incubators, refrigerators,
• Electrical power, water supply,
telecommunications ports, and other features
necessary to support such equipment should
be part of the design.
• Special Laboratory Bench Space
• Anearobic Bacteriology
• - The anaerobic bacteriology needed by most
hospital-based labs CMLs can be accomodated
by countertop jars.
-For larger labs., sufficient space should be
allocated to accommodate an anearobic
chamber and gas supply.
• Mycobacteriology, Mycology, and Virology:
-The primary design requirement for
mycobacteriology, mycology, and virology labs
• -refrigerated centrifuges, special diagnostic
equipment, and incubators required to hold
specimens at various temps.
• -equipment for performing tissue cultures
may also be needed.
• -Mycology and mycobacteriology must have a
certified class II BSC.
• Biosafety Level 2 and 3 Conditions:
• -Most routine CM procedures can be
performed safely under BSL2 conditions.
• - Even though BSL2 conditions can be met
• - It is strongly recommended that all specimen
processing be done in a class II BSC
-Isolates or cultures that are known to contain
high-risk microorganisms should be processed
only in a class II BSC, particularly fungal and
mycobacteriology cultures.
- Positive blood and CSF cultures should also
be processed in a class II BSC.
-Adequate space, power, and ventilation
should be provided for each BSC.
• Requirements for BSL2:
- Lab personnel should have specific training in
handling pathogenic agents.
-Personnel be directed by competent scientists
-Lab access is limited when work is being
conducted
-Extreme precautions be taken in handling
contaminated sharp items,
-Procedures likely to generate infectious
aerosols or splashes are conducted in either a
class II BSC or other physical containment
• Requirement for BSL3
• BSL3 conditions include all of BSL2 plus special
facilities, equipment, and procedures for
handling pathogenic and potentially lethal
agents.
-Limited lab access
- written policies and procedures for handling
agents
- adequate training, proficiency, and
competency for handling agents
-use of BSCII for handling highly infectious
• The facility requirement include:
- a separate area with access through two sets
of self-closing doors
- sealed floors, walls, and ceilings to facilitate
decontamination
- a waste disposal system that is available
within the area
-a ducted air system that draws clean air from
outside the area, with all of the exhaust air
discharged to the outside
- the use of filters in the exhaust of BSCs, in
• Molecular Microbiology
• Planning for such a lab should optimize
workspace flexibility since this technology is
rapidly changing .
-It will also depend if the lab staff plans to
perform FDA –approved and commercially
available kit-based assays or commercially
amplification kit-based assays.
-Development and performance of in-house
methods that involve amplifying NA require a
more elaborate lab.
• The three areas include :
-area for reagent preparation.
-area for specimen preparation
- area for amplification.
- Analysis of DNA and RNA requires an
assortment of basic and specialized
equipment including deionized water, wet ice,
dry ice, autoclave, fume hood, storage cabinet
for solvents and flammables and dark room.
-The reagent prep area is the cleanest work
area and can be located in a separate room.
 -never be brought into this area. Dedicated
equipment should be used to prevent
contamination.
-Reagents should be stored in a refrigerator free
of DNA, and disposable items like tubes, tips,
and gloves should also be stored in a manner
to prevent decontamination.
-Staff should wear clean lab coats and clean
gloves and should leave transportable items
like pens, tapes and scissors in this area.
• In the specimen prep area, tubes containing
 - Specimen prep activities can be performed in
the open lab on a bench, but might also be
performed in an enclosed space such as
benchtop hood.
- After NA is added to the reaction tube in this
area, the tubes are sealed and placed in the
thermocycler for amplification. When
removed, the tubes should be taken
unopened to the third area for product
detection.
• The third area is the most contaminated of the
three work areas.
• -Some Labs have opted for separate areas for
darkroom/gel electrophoresis room, a room
for processing of radioactive samples, and a
room for other functions.
• Work Flow
- Design of a Lab should be such that specimen
flow is unidirectional.
- Specimen receipt, plating, incubation, isolate
identification, AST, and result reporting.
• Lab storage
- Adequate storage space should be provided.
 Isolation and Identification of medically
important organisms
Collection, transport and examination of
clinical specimens
• Usefulness of analytical procedures performed
on clinical specimens is directly influenced by
the quality of specimens.
• Quality is in turn, largely a product of the
nature of the clinical specimen (i.e., it is
representative of the infectious disease
problem and how it was collected) and how it
was collected. Emphasis should be on safety,
selection, collection, transportation,
• -It is the key to accurate lab diagnosis that
directly affects patient care and patient
outcome
• -influences therapeutic decisions, it affects
hospital infection control, patient length of
stay, and overall hospital costs
• -It plays a major role in Lab costs, and
influences lab efficiency.
• - Every Lab must therefore develop a rational,
sound, and relevant specimen management
policy and enforce it as strictly as possible.
• SAFETY
-Biosafety at the lab bench is of primary
concern to lab staff.
- Health care workers may be unaware of the
potential etiologic agent(s) residing in the
specimen being transported to the lab
- Policies to protect staff from accidental
exposure must be in place.
- Wear gloves, gowns, and, where appropriate,
masks and/or goggles when collecting
specimens.
-bag with a separate compartment for
paperwork
-Never transport syringes with needles to the
Lab . Instead, transfer the contents to a sterile
tube or remove the needle with a protective
device, recap the syringe, and place it in a
sealable, leak-proof plastic bag
-Do not transport leaking specimen containers
to the Lab or process them. Notify the
physician or the responsible nurse and explain
the potential compromised nature of the
• Selection and Collection of The Specimen
- before a specimen is collected for analysis,
the specimen or collection site must be
selected and must represent a location of
active disease.
-Some of the common sites of infection where
ready sources of contamination reside include
the bladder, where urethral orgs and those of
the perineum may easily contaminate the
urine specimen; blood, often contaminated by
commensal flora from the venipuncture site;
the endometrium, which may contain vaginal
• General specimen selection and collection
guidelines should include the following:
- Avoid contamination from indigenous flora,
when –ever possible, to ensure a sample
representative of the infectious process.
Specimens from many sites of infection may
contain an etiologic agent that would be
considered part of the normal flora in a
healthy host. This ‘background noise’ of
normal flora could interfere with the
interpretation of culture results as well as
overgrow and obscure the true agent.
• - Optimize the capture of anaerobes from
specimens by using the proper precautions,
procedures, and supplies; biopsy or needle
aspirates are the specimens of choice, while
anaerobic swabs are the least desirable. Never
refrigerate specimens submitted for anaerobic
culture but, rather, maintain them at RT.
- Collect adequate volumes; insufficient
material may yield false-negative results.
- Place the specimen in a container designed to
promote the survival of suspected agents and
to eliminate leakage and potential safety
• Transportation
- All specimens must be promptly transported
to the Lab., preferably within 2 hrs. If
processing is delayed, specimens collected for
the detection of bacterial agents may be
stored under specified conditions.
-In general, do not store specimens for
bacterial culture for more than 24hrs. Viruses
however, usually remain stable for 2 to 3 days
at 4 degrees cent.
-Optimal transport of clinical specimens,
-Environmentally sensitive orgs include Shigella
spp (which should be processed immediately),
Neisseria gonorrhoeae. N. meningitidis, and
Haemophilus influenzae (which is sensitive to
cold temps). Never refrigerate spinal fluid,
genital, eye, or internal ear specimens.
-Transportation of clinical specimens and
transportation of infectious substances from
one health care facility or Lab to another,
regardless of the distance, requires strict
attention to specimen packaging and labeling
instructions. Materials for transport must be
• Bacteria and Fungal Specimen Transport.
-The potential etiologic agent suspected in the
patient dictates the specific collection method
and transport system that will support the
viability of the agent.
-Specimens for fungal cultures should not be
collected with a swab because of the potential
interference of the swab fibers with direct
microscopic examination of the specimen.
- Most specimen containers must be sterile
since the presence of contaminating flora
- Sterile petri dishes or specific envelopes can
be used to transport hair, skin, or nail
scrapings to the mycology Lab.
-Commercial transport devices for N.
gonorrhoeae such as the JEMBEC (John E.
Martin biological environmental chamber)
system with CO2 tablets may provide better
results than CO2-containing bottles, especially
for transport by courier. The bottles may not
have consistent amounts of CO2 and improper
manipulation during inoculation will cause a
loss of the atmosphere.
• Virus, Rickettsia, Chlamydia, and Mycoplasma
Transport
- The methods and media used for the
transport of bacteria are inappropriate for the
transport of viruses and chlamydiae.
-Viral transport media (VTM) prevent drying,
maintain viral viability during transport, and
prevent the overgrowth of contaminating
bacteria.
- Many of the formulations contain either
Eagle’s minimum essential medium or Hanks’
balanced salt solution along with fetal bovine
- There is little evidence in the literature that
one VTM is better than another. However, in
all cases in which a specimen is submitted for
viral analysis, the specimen should be selected
and collected in a manner appropriate for the
target organ.
- Liquid based transport systems contain a
protein (BSA, gelatin, or FBS) and a
combination of antimicrobial agents in a
buffered solution.
-If specimens arrive in the Lab after having been
• Specimen Acceptability or Rejection Criteria.
-At times , specimens arriving in the Lab may
have been improperly selected, collected, or
transported. This is equivalent to the
specimen being out of control.
-Processing and reporting of these specimens
to the physicians may provide misleading
information that can lead to misdiagnosis and
inappropriate therapy.
-Consequently, the Lab must adhere to a strict
policy of specimen acceptance and rejection.
• 1. No label. Do not process. Contact the
submitting physician or nurse. For specimens
obtained by noninvasive means (urine,
sputum, or throat swab specimens), have a
new specimen submitted. For specimen
obtained by invasive procedures (needle
aspirates, body fluids, or tissue), process the
specimen only after consulting with the
physician who obtained the specimen. Note
the problem on the report, and document the
corrective action taken.
• 2. Prolonged transport. Do not process. Alert
• 3. Improper or leaking container. Do not
process. Call the submitter and request a
repeat specimen. Note the problem on the
patient’s report and the corrective action
taken.
• 4. Specimen unsuitable for request (eg
request for anaerobic culture for a specimen
transported aerobically). Do not process .
Contact the submitter, clarify the test request,
and indicate the discrepancy . Request a
proper specimen for the test requested.
• 5. Duplicate specimen on the same day for the
-There may be instances in which a given
specimen must be processed even though its
quality is compromised, eg, a difficult or
unusual case, and then only after a consult
between the patient’s physicain and the Lab
director.
- Sterile body fluids may be submitted from
patient’s with serious or life- threatening
illnesses and must be handled quickly and
appropriately .
-The decision of whether to centrifuge the fluid
and culture the specimen on agar media or in
 Throat and Nasopharyngeal specimens

• Aid in diagnosis of infectious disease of URT.

• Obtained with Sterile Calcium alginate swab.

• Preserve viability of organisms by either

directly inoculating on culture medium or
place in a transport medium.
 Sputum

 Secretion of the LRT.

 May be contaminated by oral microflora.

 May be collected by expectoration, nasotracheal or

orotracheal secretion.

 Best collected in the morning because of availability.

 Reduce oral flora contamination by washing the mouth.

 Should be collected in a large-mouth sterile cup with a

tight-fitting lid to prevent spillage and contaminate.
 Ear and Eye Specimens

 For ENT investigations

 Eye specimens are collected using sterile swabs and put

in transport medium.

 Tears flushing activity and antimicrobial substances

may destroy bacteria not processed immediately.

 To collect ear specimens, the external surfaces should

be decontaminated with ROH.

 Use sterile swab, or pus can be aspirated into a sterile

syringe.
 Stool and rectal swabs

• Collect feces specimens in the morning, i.e.

first stool of the day because of availability.

• Most valuable when collected early in the

course of the day.

• Avoid contamination with urine; but does not

require sterile containers.

• Most intestinal pathogens sensitive to

 Cont…..

• Intestinal protozoa may alter their

characteristic forms in specimens not
examined soon after collection.

• Some organisms are fastidious and may lose

viability in feces. Rectal swabs may offer a
useful alternative.

• Isolation of pathogens such as viruses from

feces usually requires shipping specimen to the
nearest viral labs.
 Genital tract specimens
 Venereal disease and genital infections investigation

 Cervical-vaginal swabs, urethral exudates, fluids from

genital lesion.

 For vagina swabs, care should be taken to avoid

contamination with fecal organisms from the rectum.

 Place swabs in tubes of sterile saline.

 If trichomoniasis is suspected, the specimen should be

microscopically examined within 15 mins since they
quickly lose motility making identification difficult.

 Blood is employed for HIV and primary syphilis

infection.
 Urine collection
 For UTI determination.

 Contamination of properly collected urine is inevitable; fewer than

10.000 organisms/ml is considered normal.

 When concentration of Gram –ve bacilli exceeds 100,000 cells/mL,

they are declared etiologic.

 Urine is an excellent culture medium, specimens should not be

allowed to stand at RT for > 30 mins; overgrowth may lead to
misdiagnosis.

 If delay is unavoidable, specimen should be stored at 4oC for no

longer than 6hrs.

 To avoid contamination, voided urine should be collected by “clean

catch” or midstream-catch technique.
 Specimens from skin lesions

• Swab draining rashes or purulent lesions after

cleaning with alcohol or iodine to remove the
pus.

• Aspirate closed lesions directly into sterile

syringes.
 Specimens from wounds

• Obtain swab or aspirates depending on the

nature of wound.

• Infected superficial wounds always contain

aerobic organisms.

• Deep wounds or abscesses often support the

growth of obligate anaerobes: culture
 Biopsy material

• Biopsy specimens are commonly fixed in

formalin before examined microscopically.

• Killed by formalin, therefore useless for

culture.

• Fresh specimen for culture should not be put in

formalin.

• Biopsy material has to be aseptically ground

 Cerebrospinal fluid

• CSF is normally sterile if properly collected.

• Analysis should be done ASAP to prevent

fatality.

• Morphology of organisms necessary to guide

 Blood specimen

 Decontaminate vein puncture site before collection; use

alcohol, iodine or acetone.

 Apply a tourniquet and draw the specimen aseptically

into a syringe.

 Draw enough blood (5-10ml) to inoculate both aerobic

and anaerobic culture media.

 Additional samples may be collected for quantitative

plate counts, microscopic examination, and differential
characterization of blood stained cells.

 Blood could also be collected for immunological

analysis to detect microbe- specific antibodies.
• LABORATORY EXAMINATION OF CLINICAL
SPECIMENS.
-In dealing with an infection, one often is
immediately faced with species identification in
order to prescribe effective treatment.
-The typing techniques that have evolved for such
discrimination must be rapid and accurate
-These techniques usually focus on phenotypic
characteristics and rarely provide the resolution
necessary to obtain measurements of genetic
relatedness between isolates of the same species or
subspecies.
-To accurately identify the origin of a
nosocomial infection, transmission of a
disease between individuals, the emergence
of a new hypervirulent or drug-resistant
strain, the microevolution of a commensal or
infecting strain, and the general population
structure of a pathogen, one must move from
species typing to strain or and sub-strain
typing.
- Prior to the development of DNA-based
techniques for assessing genetic relatedness ,
scientist relied on biotyping techniques, which
Several procedures are critical to the diagnosis of
infectious disease:
Microscopy
• Preliminary microscopic examination of
stained smears or wet mounts can provide
early indications of types of pathogens.
• Wet mounts consist of clinical material in a
drop of saline to determine cellular
composition, morphology and motility.
• Gram’s stain of sputum, CSF, or blood can
 Cont…

• Acid-fast smears can alert of TB.

• Direct dark – field examination of fluid from

suspected syphilis chancres can often reveal
the presence of motile spirochetes.

• Other microscopic techniques could be applied

after culture and staining (Gram, acid-fast,
spore, capsule, etc.).

• Microscopic techniques: Bright field, dark

 Culture

• Culture undoubtedly constitutes the gold

standard for the identification of most
microorganisms.

• Different specimens cultured on appropriate

media (stool, urine, blood, etc)

• It also allows the susceptibility to antibiotics

 Cont…

• Some specimens may yield positive cultures

after delays.

• Blood may take up to 3 weeks; TB, 8 weeks

and some fungal cultures up to a week.

• May use streak or pour plate techniques.

 Biochemical characterization (Biotyping)

• Wide array of tests could be used to

differentiate between microbes that are
morphologically similar.
• Sugar fermentations, gelatin utilization, VP
reaction etc. could be used.
• API (Biomerieux) kit very useful, Vitek
The VITEK 2 is an automated microbiology system utilizing
growth-based technology :VITEK 2 compact,
VITEK 2, and VITEK 2 XL used for identification based on
colorimetric reagent cards that are incubated and
interpreted automatically.
• BIOMIC V3: can read anything from 96-well
• BIOMIC V3 is an open system utilizing digital
imaging to automate the reading and
interpretation of clinical microbiology tests
and QC from various manufacturers.
• Systems are customized with optional
modules including antibiotic susceptibility,
organism identification, 96-well microtiter*,
and colony counting.
 Antibiograms

• Determination of antibiotic susceptibility.

• Resultant Antibiograms used as guidelines for

empiric treatment.

• Kirby – Bauer disc diffusion [Should conform

to Clinical and Laboratory Standard Institute
(CLSI)].
 Kirby – Bauer disc diffusion Method

 Prepare bacteria inoculums from subcultures

 Emulsify 4-5 colonies of the isolate in 3ml sterile

normal saline

 Adjust turbidity to 1.5x108 CFU/ML (corresponding to

0.5 McFarland standards).

 Dip a sterile cotton swab into the standardized bacteria

suspension and evenly inoculate agar plates; allow to
dry for 10-15 minutes.

 Thereafter, place the antibiotic dics on the plates and

press gently to ensure complete contact with agar.
 Cont…
 A distance of at least 15mm should be maintained
from the edges of the plates to prevent
overlapping of inhibition zones.

 Incubate plates at 37OC for about 24-48 hours.

 Examine them and measure the diameter of zone

of inhibition.

 Include a control strain of a known organism in

all the experiments to determine susceptibility or
resistance. This could be ATCC or NCTC
 Determination of Minimum Inhibitory Concentration
(MIC)
 The MIC can be determined by the agar dilution method

 Prepare a stock of each antibiotic by dissolving the powder

in 10ml of sterile phosphate buffered solution (pH 7.2).

 Carry out a Two-fold serial dilution of each antibiotic in

PBS.

 Obtain an appropriate concentration for the range of

antibiotics to be used.

 For example, prepare a series of 13.5ml of the base medium

(Brain Hearth Infusion Agar) and add 1.5ml of the serially
diluted antibiotic to the medium.
 Cont…
• Pour into sterile petri dishes and allow to
solidify.

• Prepare inoculum of organism to be used as

described above (0.5 McFarland).

• Inoculate the plates and incubate appropriately.

• After incubation, read the MIC value as the

lowest concentration of the antibiotic that
inhibited bacteria growth (no visible growth).
 MBC Determination

• MBC is determined by subculturing the MIC

assay tubes onto fresh agar plates.

• The highest dilution that yielded no single

bacterial colony on the medium is taken as the
MBC.
 Serotyping
• Could either detect antigens or antibodies in
patients’ sample.

• Information offers indirect indications of the

etiology of disease before culture is performed.

• Very useful when dealing with fastidious or

delicate pathogens.

• Techniques using precipitation, agglutination,

fluorescent or ELISA based approaches etc.
• The western blot, sometimes called the
protein immunoblot is a widely used
analytical technique used in molecular
biology, immunogenetics and other molecular
biology disciplines to detect specific proteins
in a sample of tissue homogenate or extract.
• Synthetic animal-derived antibodies are
created that react with a specific target
protein.
• The sample to be tested is prepared and put
together with these antibodies on a
membrane – if the specific protein sought for
• Methods developed to complement Serology
• Pulsed-field gel electrophoresis
• Multilocus enzyme electrophoresis
• Variable number-of- tandem repeats
• Multilocus sequence typing
• However, genes encoding serological relevant
Ags. vary and prone to recombination
• Therefore valuable to develop a valuable mol
identification scheme to include serotype and
genotype to complement serology.
 Practical Applications of Immunology
1. Vaccines
2. Diagnostic tests

Vaccines

• Chinese were first to “vaccinate”: variolation

• -Ground up small pox scabs, rub into wound

• -Some people got mildly ill and then were

immune to small pox, some died.

Jenner developed first Western vaccine:

 Cont…
• -observed that milk maids that got cow pox
never got small pox.

• -He inoculated people with cow pox to prevent

small pox infection

• -Set stage for vaccine development. (vacca =

cow: name given by Pasteur later)

• vaccine = suspension of organisms or fraction

of organisms used to induce immunity.
 Mechanism of action:

• -Exposure (injection) induces primary immune

response:

• -Antibodies and long term memory cells are

formed; slow, takes 1-2 weeks.

• -Natural exposure later induces secondary

immune response:

• -Memory cells stimulated to act

 Cont…

• Rapid and intense response

• -Pathogen destroyed before it causes Disease.

• Herd immunity = having enough of the

population vaccinated to prevent spread of
disease
 Diagnostic Immunology
Diagnostic immunology is the future of medical
diagnosis for infectious disease.
• -Use purified antibody solutions (antiserum) to
diagnose disease

• -Diagnostic antibodies can be produced to

detect particular microbes:
• 1. Inject animal with microbe or antigenic
fragments
• 2. Allow immune response (1-2 weeks)
• 3. Harvest blood
 Agglutination Reactions

• -To detect particulate antigens in solution

• -Antibodies cause clumping (agglutination) of

their specific antigens

• (e.g. Hemagglutination for blood typing:

detects surface antigens on RBCs).
 Direct agglutination tests

• -To detect if patient has antibodies to particular

Antigen (antibodies present = exposure or
infection by the agent).

• -known infectious agents are bound to a

microtiter dish

• -Patient serum is added

 Indirect agglutination tests

Indirect = uses latex particles

Two ways:
• 1. -known antigen is bound to latex particles
-Assay for patient sample for antibody.

• 2. -known antibodies are bound to latex

particles
-Assay for antigenic agent
 Fluorescent Antibody Labeling

• -Identify or visualize microbes in clinical

specimens

• -Use antibody chemically linked to florescent

dye that is visible with UV light

• -Can be used to tag and sort cells (e.g.

stemcells, cancer cells, male/female sperm
etc.)
 ELISA
Enzyme Linked Immunosorbant Assay

• -Used to detect either antibodies or antigens in

patient sample

• -Performed in microtiter plate

• -Positive reaction produces color change

• -Process can be automated: computer readout

of many samples at once
 Direct ELISA (“Sandwich ELISA”)

• A sandwich ELISA measures antigen between

two layers of antibodies (capture and
detection antibody). The target antigen must
contain at least two antigenic sites capable of
binding to antibodies.
-Antibody bound to dish.
-Monoclonal or polyclonal antibodies can be
used as the capture and detection antibodies
in sandwich ELISA systems.
-Assay for presence of antigen in sample
 Indirect ELISA

• -Antigen bound to dish

• -Assay for presence of antibody in sample

 Western Blot

-Often used to confirm ELISA positive result

-Blot assays both size of protein antigen and

specific reaction with antibody

-Size confirmation proves/disproves possible

cross reaction (false positive)
 Method:

• 1) Proteins collected from patient sample and

separated by size on gel electrophoresis.
Electric field drives proteins through gel:
large stay near top, small move toward
bottom

• 2) Separated proteins are transferred (blotted)

to a nylon membrane
 Cont…

• 4) Antibodies in antiserum specifically bind to

their epitope

• 5) A colored substrate is added: reacts with the

Fc region of bound antibodies thus coloring
location of antigen with bound antibody

• 6) Pathogen is confirmed by: (i) binding of

 Molecular typing
• Isolating Genomic DNA from Gram negative bacteria
• Principle of the procedure

– The Wizard Genomic DNA Purification Kit is based on a four step

process. The first step in the purification procedure lyses the
cells and the nuclei. The cellular proteins are then removed by a
salt precipitation step, which precipitates the proteins but
leaves the high molecular weight genomic DNA in solution.
Finally, the genomic DNA is concentrated and desalted by
isopropanol precipitation
• Targets nucleic acids (DNA/RNA), preferably
DNA since unique as fingerprints.

• Very useful in cases where the organism can

not be cultured.

• DNA probe can be used to target unknown

organisms with identical or different DNA
 Cont…
• PCR – based techniques such as conventional
PCR, RT – PCR, RFLP – PCR, RAPD, qPCR
are great tools.
• Ingredient n reactions
– Prepare a master mix for PCR. Aliquot out PCR
– Mix into reactions tubes.
– Buffer KCl 5
– Magnesium Chloride (50mM)
– Universal Primer 1 (10mM)
– Universal Primer 2 (10mM)
– dNTPs (5mM)
– Taq Polymerase
– PCR Water
• Explanation of chemistry for PCR
– The PCR buffer provides the optimal salt conditions for the Taq polymerase.
– MgCl2 is used to stabilise the DNA before, during and after synthesis.
– The concentration of MgCl2 has been optimised and is specific to each PCR
reaction.
– Clinical PCRs should have negative and blank controls, the negative has water
added at the same time as DNA and blank is not opened outside the clean lab.
– Bands in either control suggest contamination added at the same time as DNA
and blank is not opened outside the clean lab.
• Bands in either control suggest contamination
at either stage and can help to identify
sources of contamination.
• The PCR cycles consist of temperatures that
are optimal for DNA strand separation (95° C),
annealing of primers to template (55° C), and
extension by Taq polymerase (72° C).
• Explanation of chemistry for electrophoresis
– Electrophoresis of DNA through agarose is used to separate DNA on
the basis of size and charge, the negatively charged DNA migrates
towards the positive electrode.
– The matrix of the agarose provides a resistance to the migration so
that different sizes are pulled different distances by the same charge,
according to their size.
– The percentage of the gel should be appropriate for the size of the
DNA in the sample so that the larger the fragment, the lower the
percentage required.
– The PCR product will migrate to a position in the gel according to its
size, the markers in the ladder will separate and comparison to the
ladder allows a size estimation of the PCR product.
• Ethidium bromide is incorporated into the
agarose gel.
• Ethidium bromide binds to the minor groove
of the DNA double helix. N.B. It will bind to
your DNA too and is mutagenic so do not
touch the gel without gloves on.
• Ethidium bromide is incorporated into the PCR
product as the band migrates through the gel,
and will
• glow in the UV, hence making the bank visible.
• Sequencing of PCR products important for
proper characterization.
• Explanation of chemistry for clean-up
– The clean up column works with the same
principle as the DNA extraction, except
– the column is optimised fro fragments of DNA
rather than whole DNA. This clean up
– process is to remove Taq polymerase and dNTPs
from the reaction as they interfere
– with the sequencing reaction.
• Explanation of sequencing chemistry
– The sequencing reactions work like a PCR reaction
except that the ready reaction mix contains a
mixture of dNTPs and dNTPs with fluorescent dye
terminators.
– The dye terminators stop the elongation of the
DNA strand and are fluorescently labelled so that
the terminal nucleotide fluoresces with a colour
according to its base, i.e. T is red.
– This occurs on a random basis, so that there will
be a fluorescing terminal
• residue corresponding to every base in the sequence, as illustrated.
• The sequencing reaction is then electrophoresed on a machine that uses a
laser to read the fluorescence of each dye as it passes, thus determining
the sequence.
• ATGATCCG*
• ATGATCC*
• ATGATC*
• ATGAT*
• ATGA*

• ATG*
• AT*
• A*
• Next-Generation DNA Sequencing
• Next-generation sequencing (NGS), also
known as high-throughput sequencing, is the
catch-all term used to describe a number of
different modern sequencing technologies
including:
• Illumina (Solexa) sequencing
• Roche 454 sequencing
• Ion torrent: Proton / PGM sequencing
• SOLiD sequencing
• These recent technologies allow us to
sequence DNA and RNA much more quickly
and cheaply than the previously used Sanger
sequencing, and as such have revolutionised
the study of genomics and molecular biology.
• In situ Hybridization
– Most sensitive and specific means of identifying the location of
genes on chromosome.
– Requires NA probes labeled by incorporation of nucleotides
modified with molecules such as biotin.
– Visualization is achieved using Abs conjugated to fluorochromes.
– Major advantage is that radioactivity not required; rapid;
amenable to computerized storage and manipulation; sensitive;
gives accurate signal localization; allows simultaneous analysis
of 2 or more fluorochromes, and provides quan and spatial
distribution of the signal.
• Microarrays
– Collection of microscopic features, most
commonly DNA which can be probed with target
molecules to produce either quantitative effects
such as gene expression, or qualitative such as
diagnostic data.
– Various types do exist: protein, DNA etc.
– Microarrays can be distinguished based on
characteristics such as nature of the probe, the
solid-surface support used and the specific
method used for addressing and/or target
detection