2021 laboratory diagnosis of infectious diseases dr. ihsan alsaimary university of basrah - college of medicine- DEPARTMENT OF MICROBIOLOGY POBOX 696 ASHAR BASRAH 42001 IRAQ

1. LABORATORY DIAGNOSIS
OF INFECTIOUS DISEASES
Prof. Dr.Ihsan Edan Alsaimary
Department Of Microbiology,
College Of Medicine-university Of Basrah

2. COMMUNICATION BETWEEN PHYSICIAN AND
LABORATORY
The techniques used to characterize infectious
depending on the clinical syndrome and the type
of infectious agent.
Because no single test will permit isolation or
characterization of all potential pathogens,
clinical information is much more important for
diagnostic microbiology than it is for clinical
chemistry or hematology.

3. Diagnosis of infectious diseases
The proper diagnosis of an infectious disease
requires :
(1) patient history.
(2) physical examination of the patient.
(3) carefully evaluating the patient’s signs and
symptoms.
(4) conducting appropriate laboratory tests and other
methods.
(5) proper selection, collection, and transport of
appropriate clinical specimens.

4. Guidelines for the collection and
transportation of specimens must
emphasize two important aspects
I-Collection of the specimen before the administration
of antimicrobial agents.
II-Prevention of contamination of the specimen with
externally present organisms or normal flora of the
body.

5. The Three Components of Specimen
Quality
Component Explanation
Proper selection of the
specimen
The specimen must be the appropriate type to diagnose the
infectious disease suspected by the clinician.
Proper collection of the
specimen
Proper collection eliminates or minimizes contamination
of the specimen with microflora. Sometimes special
collection devices are required.
Proper transport of the
specimen
The specimen Transport let viable and/or preserves agent
morphology (e.g., rapid transport, sometimes by packing
the container in ice or by using a preservative)

6. PROPER SELECTION, COLLECTION AND
TRANSPORT OF CLINICAL SPECIMENS
1-The specimen must
be properly selected.
Collect the appropriate type of specimen for diagnosis of the
infectious disease.
2-The specimen must
be properly and
carefully collected.
1-From a site of pathogen is most likely to be found.
2-Before antimicrobial therapy has begun
3-The acute stage is the best
4-Avoid harming the patient and causing discomfort.
5-Sufficient quantity.
6-All specimens should be placed or collected into a sterile
container
3-Specimens must be
properly transported
to the lab
Specimens should be protected from heat and cold to ensure
represent the number and types of organisms present at the time
of collection.

7. QUALITY ASSURANCE AND QUALITY CONTROL IN THE
CLINICAL MICROBIOLOGY LABORATORY
A-Quality Assurance (QA)
B-Quality Control (QC)

8. QualityAssurance (QA)
It is continuously
identify, monitor,
evaluate, and
improve the
reliability

9. Quality Control (QC)
Is designed to monitor the accuracy, reliability, and
reproducibility of all tests performed within the laboratory and
to identify and correct any problems that exist.
Components of a CML’s QC program include the following:
1-Standard operating 2-Test verification.
procedures manual.
3-Test methods and
procedures.
The procedures written for every
aspect of CML work.
use methods for most accurate
The QC program must monitor, document, and
evaluate all aspects of every test procedure that is
performed in the CML.

10. 4-Media, reagents, staining solutions, antisera.
5-Equipment and instruments.
6-Records and reports. 7-Proficiency testing.
Avoid expiration dates
maintained and monitored of
equipment for performance.
QC measures and
documented
positive and negative controls
used for every test procedure

11. Criteria for rejection of specimens
i-Missing or inadequate identification.
ii-Insufficient quantity.
iii-collected in an inappropriate container.
iv-Contamination suspected.
v-Inappropriate transport or storage.
vi-Unknown time delay.
vii-Haemolysed blood sample.

12. CLINICAL SPECIMENS FROM VARIOUS ANATOMICAL
SITES AND ORGAN SYSTEMS
Circulatory System
Skin, Abscess, and Wound Specimens
Eyes and Ears
Respiratory System
Central Nervous System
Urinary Tract
Genital Tract
Oral Cavity
Gastrointestinal Tract
Body Fluids

13. Circulatory System
Blood
Blood is usually sterile.
Bacteremia:presence of bacteria in the
bloodstream.
Septicemia condition: bacteria product or any
toxic substance in blood.
The severe types of septicemia are caused
by Gram-negative bacilli that release
endotoxin from their cell walls, it can
induce fever and septic shock, which can be
fatal.

14. phlebotomy

17. COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Most important aspect of laboratory
medicine
Insufficient quantity
Contamination
Improper transport media
Delay in transportation
Inappropriate storage

18. Preanalytical
Phase
Analytical
phase
Post
analytical
phase
Diagnostic
cycle

19. THE DIAGNOSTIC CYCLE
PRE-ANALYTICAL PHASE
Visit
Examination
& Provisional
diagnosis
Collection
of sample
Identity
Requisition
Transport
Records

20. ANALYTICAL PHASE
Macroscopy Microscopy Culture
Biochemical
identification
Serology Molecular
Analysis

21. POST-ANALYTICAL PHASE
Reporting of
Identification &
Antibiotic sensitivity
Treatment

23. Specimen
Collection &
Transport
Collection
from actual
site with
minimum
contamination
Optimal
time of
collection
Sufficient
quantity of
specimen
Appropriate
collection
device,
container &
culture media
Proper
labelling of
containers
Obtain
samples
before
starting
antibiotics

24. BLOOD CULTURES
• One blood culture usually consists
of blood from a single
venepuncture inoculated into two
separate bottles (one aerobic,
one anaerobic)
• Optimal blood-to-broth ratios are
1:5 to 1:10
.
• Sodium polyanethol sulfonate
(SPS)-
0.025to 0.05%
-
na sa dedda si
citycogahpitna na ,tnalugaocitna
setavitcani taht tnega
gnizilartuen a dna ,tnemelpmoc
ynam fi stceffe tibihni ot tnega
lairetcabitna dna laiborcimitna
factors in blood.

25. • Recommended total volume and numbers of blood
cultures
Syringe needed Aerobic bottle Anaerobic bottle
Adult 20 ml 10 ml 10 ml
Pediatric 20 ml 2.5 -10 ml 2.5 -10 ml
Infant 3 ml 0.5 -1 ml 0.5-1 ml
Adult
(low volume) All None
• Timing of blood cultures
 Before or during fever spike
 Acute sepsis or another condition (osteomyelitis,
meningitis, pneumonia or pyelonephritis): two blood
cultures of maximum volume consecutively from different
anatomical sites before starting therapy

26.  Pyrexia of Unknown Origin, Subacute Bacterial Endocarditis or
other continuous bacteraemia or fungemia: maximum of three
blood cultures with maximum volume
 Patients on antimicrobial therapy : Sample is drawn when
antimicrobial agents are at their lowest concentration.
• Skin antisepsis and collection of blood from venepuncture
 A different venepuncture site for each blood culture is selected.
 If blood for culture is required to be drawn through a port in an
indwelling catheter, the second culture must be from a peripheral
site.
 Vigrous cleaning is done with 70% isopropyl or ethyl alcohol.
Allowed to dry.
 Swab or wipe concentric circles of 1 to 2% tincture of iodine,
moving outward from centre of the site.
 Allow the iodine to dry and avoid touching the site.

27. Skin, Abscess, and
Wound Specimens
skin is a type of host
defense mechanism, serving as a physical barrier.
•
Sebaceous glands: Glands in the dermis that usually open
into hair follicles and secrete an oily substance known as
sebum.
Folliculitis: Inflammation of a hair follicle
Sty (or stye): Inflammation of a sebaceous
gland that opens into a follicle of an eyelash.
Furuncle: A localized pyogenic (pus-producing) infection
of the skin, usually resulting from folliculitis; also known as
a boil.

28. Obtaining specimens from the skin:
1-For pustules :
The covering removed using a sterile needle, then fluid and basal cells collected.
2-For petechiae :
specimen is collected by vigorously scraping the outer margin of the lesion.
3-Collecting abscess specimens:
the abscess contents are aspirated using a needle and syringe.
4-Collecting wound specimens:
specimen should be an aspirate, or a sample taken from
the advancing margin of the lesion.

29. Eyes and Ears
Eyes
infectious diseases of the eye include the following:
Conjunctivitis: An infection or inflammation of the conjunctiva.
Keratitis: An infection or inflammation
of the cornea.

30. Keratoconjunctivitis: An infection that
involves both the cornea and conjunctiva.
specimen as conjunctival swab, corneal
scraping, or aspirate.
Using separate sterile swabs, sample the
conjunctiva of both eyes, even if only one eye is
infected.

31. Ears
There are three pathways for pathogens to enter
the ear:
(1) Eustachian tube, from
the throat and nasopharynx.
(2) from the external ear.
(3) via blood or lymph.
otitis media Infection of the middle ear
otitis externa infection of the outer ear canal.
specimen collected by swab of external ear canal,
fluid aspirated from the middle ear,

32.  First the aerobic bottle is inoculated and then the anaerobic bottle.
• Collection of blood from intravascular catheters
 Comparison of cultures that are drawn through an indwelling
intravenous catheter and through a peripheral site may be useful
for diagnosis of catheter related sepsis.
 Bottles are labelled with patient name, site, date and time of draw.
 Using two separate alcohol preps, catheter hub connection is
scrubbed for 15 sec with 70% alcohol. Air dry.
 Wear gloves, disconnect tubing or cap of catheter and attach
syringe to collect discard blood ( 3ml for adults and 0.2ml for
pediatric patients), which is not used for culture.
 Using a new syringe, blood for culture is collected through the
hub.
Quickly reconnect tubing.

33. • Specimen Transport
 Blood cultures should not be refrigerated. They are generally
held at room temperature until processed, for a maximum of
4 h.
• Rejection Criteria
 Blood cultures that are received unlabelled.
 If the tube or bottle is cracked or broken.
 Labelled blood cultures are not rejected even if medium is
expired, volume or number of bottles is insufficient, or
bottles are received
>
12 h after collection.

34. Respiratory System
Specimen:
A. Nasal swabs
B. Nasopharyngeal / Pernasal swabs
C. Nasopharyngeal aspirates
D. Nasal washings
E. Throat swabs
F. Lung aspirations
G. Open lung biopsy

35. Lower Respiratory Tract Cultures
• Expectorated
sputum
• Induced sputum
 Used for Pneumocystis carinii and Mycobacterium tuberculosis
 Patient’s mouth is rinsed thoroughly with sterile water or saline.
 Using and ultrasonic nebulizer, patient is made to inhale approx. 20
to
30ml of 30% NaCl.

36. • Tracheostomy and endotracheal aspirates
 The specimen is aspirated into a sterile sputum
trap or leak proof cup.
 Tracheostomy aspirate should not be cultured
unless clinical pneumonia is present (fever and
infiltrates). Tracheostomy is followed by
colonization within 24h of insertion, and results
may not correlate with disease.
• Bronchoscopy specimens
 Include BAL samples, bronchial washings,
transbronchial biopsy specimens.
 Culture BAL samples quantitatively or
semiquantitatively for bacterial pathogens
Mucus
extractor for
ET aspirates

37. • Specimen Transport
 Specimens are collected in leakproof cups and
labelled with source of material.
 Specimens are labelled with demographic
information, date and time of collection, and site
of collection.
 Provisional diagnosis should be mentioned for
proper evaluation of the cultures.
 Appropriate tests should be requested.
Anaerobic cultures should be limited to
specimens that are not contaminated with
upper respiratory microbiota (eg. Biopsy
samples) and only when aspiration pneumonia
or similar disease is being considered.
 Specimens should be stored at 2 to 80C until
cultures can be submitted or processed.
Sputum
sample
container

38. • Rejection Criteria
 Repeat cultures at intervals of less than every 48 h.
 24h sputum collection
 Contaminated sputum and endotracheal specimens per
Gram stain rejection criteria
 Saliva
 Nasal washes and aspirates or swabs of nares
 Throat specimens
 Specimens for anaerobic culture, except transtracheal
aspirates, biopsy samples, pleural fluid or other
uncontaminated specimens.
 For specimens delayed in transit time more than 2 h without
refrigeration.

39. Central Nervous System
The nervous system is composed of the central nervous system (CNS) and the
peripheral nervous system.
It is sterilized but agent enter through trauma, blood and lymph or along the
peripheral nerves.
Glucose level indicator for infection.
Encephalitis: Inflammation of the brain.
Encephalomyelitis: Inflammation of the brain and spinal cord.
Myelitis: Inflammation of the spinal cord.
Meningitis: Inflammation of the membranes (meninges) that surround
the brain and spinal cord.
Meningoencephalitis: Inflammation of the brain and meninges.

40. Specimen
By lumbar puncture

41. Microscopic Examination :
Smears are made from the sediment of centrifuged CSF.
Gram stain :
intracellular gram-negative diplococci (meningococci)
small gram-negative rods (H influenzae or enteric gram-negative rods).
lancet-shaped gram-positive diplococci (pneumo-cocci)
Follow-Up Examination of
Cerebrospinal Fluid:
glucose level and cell count toward normal is good evidence of adequate therapy.
Culture :
Sheep blood and chocolate agar together grow almost all bacteria and fungi that
cause meningitis.

42. Urinary Tract
1-Upper UTIs : kidneys and ureters.
2-Lower UTIs : urinary bladder, the urethra, and in
males, the prostate.
Cystitis: Inflammation of the urinary bladder.
Nephritis: inflammation of the kidneys.
Pyelonephritis: inflammation of the renal parenchyma.
(the basic cellular tissue of the kidney).

43. URINE CULTURES
• Clean voided midstream urine collection
 Females- labia are held apart, vulva is washed from front to back
 Circumcised males- no preparation.
 Uncircumcised males- foreskin is retracted, glans penis is washed
 Patient is asked to collect voided urine directly into a disposable
leak proof container, instructing the patient to not halt and restart the
urine stream for a “midstream ”collection but preferably move the
container into the path of already voiding urine.
• Catheter urine
 Using a needle and syringe, urine is collected through the catheter
port lohocla htiw gninaelc retfa ,
.
 Urine obtained from a catheter bag should not be send.
• Timing of specimen collection
 Early morning specimens should be obtained whenever possible.
 Fluids should not be forced in order to have the patient void urine.

44. • Specimen Transport
 Urine should be transported to the lab after collection
or, if urine cannot be delivered within 2 h after
collection, can be refrigerated for up to 24 h.
• Rejection Criteria
 No evidence of refrigeration and the specimen is >2 h
old.
 Collection time and method of collection have not been
provided.
 24
- h urine collections
 Foley catheter tips
 Urine from the bag of a catheterized patient
 Specimens that arrive in leaky containers
 Except for suprapubic bladder aspirates, reject
specimen
requests for anaerobic culture.
Urine
sample
container

45. Genital Tract
The urethra contain microflora in males and females, special female genital region
supports the growth of many microorganisms.
Cervicitis: Inflammation of the cervix, that part of the uterus that opens into the
vagina.
Endometritis: Inflammation of the endometrium (the inner layer of the uterine
wall).
Oophoritis: Inflammation of an ovary.

46. Diseases
A.Gonorrhea
A stained smear of a urethral or a cervical exudate that shows intracellular gram-negative
diplococci strongly suggests gonorrhea. The sensitivity is about 90% for men and 50% for
women Thus.
B. Syphilis
Dark-field or immunofluorescence examination of fresh tissue fluid expressed from the
base of the chancre may reveal typical T. pallidum.

47. C. Vaginosis/Vaginitis
A-Bacterial vaginosisassociated with Gardnerella vaginalis or Mobiluncus and
parasite Trichomonas vaginalis.
The discharge :
(1) is grayish and sometimes frothy,
(2) has a pH above 4.6
(3) has an fishy odor
(4) contains “clue cells,” large epithelial cells covered with gram-negative or gram-variable rods
B-Candida albicans vaginitis is diagnosed by finding yeast or pseudohyphae in a
potassium hydroxide preparation of the vaginal discharge.

48. Oral Cavity
Dental caries: Tooth decay or cavities, Streptococcus mutans.
Gingivitis: Inflammation of the gingiva (gums).
Bacteria are isolated from oral swabs, Carefully collected
scrapings or aspirates should be transported to the CML.

49. Gastrointestinal Tract
The gastrointestinal (GI) tract consists of a long tube with many expanded areas
designed for digestion of food.
Most of the microorganisms low pH (1.5),
and are inhibited from growing in the lower
intestines by the resident microflora.

50. • Diarrhea: An abnormally frequent discharge of semi-solid or fluid fecal matter.
• Dysentery: Frequent watery stools accompanied by abdominal pain, fever, and dehydration
and may contain blood and/or mucus.
• Enteritis: Inflammation of the intestines.
• Gastritis: Inflammation of the mucosal lining of the stomach.
• Gastroenteritis: Inflammation of the mucosal linings of the stomach and intestines.

51. Body Fluids
body fluid specimens include abdominal or
peritoneal fluid, pleural or thoracentesis fluid,
synovial (joint) fluid, and amniotic fluid.
Carefully collected aspirates of amniotic and
pleural fluids are usually transported to the
CML in an anaerobic transport system.

52. Factors limiting usefulness of
microbiology investigations
Specimen should be obtained from site
of infection
Sample must be taken aseptically
Sample size must be large enough
Metabolic requirements for the
organism must be maintained during
sampling, storage, and transport.
Wrong sample
e.g. saliva instead of sputum
Delay in transport / inappropriate
storage
e.g. CSF
Overgrowth by contaminants
e.g. blood cultures
Insufficient sample / sampling error
e.g. in mycobacterial disease
Patient has received antibiotics

53. Wound and Soft tissue cultures
• Specimen should be collected prior to initiation of therapy and
only from wounds that are clinically infected or deteriorating or
that fail to heal over a long period.
• Skin or mucosal surfaces should be cleansed.
 Closed wounds and aspirates- disinfected with 2%
chlorhexidine or 70% alcohol followed by iodine solution )1 to
2% tincture of iodine or a 10% solution of povidine-iodine)
 Open wounds should be debrided ,
dna
ylhguoroht
desnir
htiw
elirets
enilas
roirp
ot
noitcelloc
.
• Viable infected tissue should be sampled, rather than superficial
debris.
• Swab collection should be avoided if aspirates or biopsy
samples can be obtained.

54. • Containers:
 Anaerobe transport vial for small tissues.
 Sterile cup for large tissues with
nonbacteriostatic
saline on a gauze pad to keep moist.
 Syringes
 Broth culture medium in small sterile snap –top
microcentrifuge tubes for FNA
 Swabs (ideally, submit two, one for Gram stain
and one for culture)
• Specimen collection
 Cold abscesses- infected material is aspirated
with needle and syringe.
 Open wounds- superficial area is cleansed with
sterile saline. Overlying debris is removed with
scalpel and swabs or sponges. Biopsy or
curette sample is collected from base or
advancing margin of lesion.
Anaerobic
specimen
collector
Swabs

55.  Pus- deepest portion of the lesion or exudate is
aspirated with a syringe and needle.
 Tissue and biopsy samples- 3
-
ot
4
-
yspoib mm
samples are collected, avoiding necrotic areas.
• Label specimen and requisition
 Demographic information of the patient.
 Type of specimen (deep tissue, superficial
tissue, decubitus, catheter site, boil, abscess,
cellulitis, aspirate, pus, drainage, surgical
incision site, etc.) should be described.
Anatomic location (arm, leg, etc.) should be
mentioned
 Collection time and date should be recorded
 Diagnosis, including cause and clinical signs of
infection should be mentioned.
 Antimicrobial therapy prior to specimen
collection should be mentioned
Aspiration of
pus using
needle &
syringe

56. • Specimen transport
 Aspirates and tissues should be delivered to the lab within 30 min
 Tissues should be kept moist to preserve organism viability
 Specimen should not be refrigerated or incubated
• Rejection Criteria
 Specimens in container with formalin
 If numerous squamous epithelial cells are present on Gram stain
 Swabs that have been delayed in transit more than 1 h if they are
not in some transport system.
 For multiple requests (AFB, fungal, bacterial and viral) but little
specimen.

57. Faecal Culture for Aerobic Pathogens
• Patient should pass stool into a clean, dry pan.
• Transfer at least 5 ml of diarrhoeal stool, 1 g of material or a walnut
sized portion of stool.
• Container:
 Clean, leak proof container with a tight fitting lid or
 Buffered glycerol saline
 Modified Cary-Blair medium
 Stool enrichment broths
 Anaerobic transport tube for C. difficile culture
• Rectal swabs
 Pass the tip of a sterile swab approx. 1 in. beyond the anal
sphincter
 Carefully rotate the swab to sample the anal crypts, and withdraw
the swab
• Timing
 Submit specimen during acute stage of infection (usually 5 to 7

58. Anaerobic Culture
• The best specimen is obtained by using a needle and syringe.
• Tissue samples and biopsy samples are also very good
specimens.
• The least desirable specimen is collected by swab
• Specimen Transport
 Tissue samples, biopsy samples, or curettings should be
placed into an anaerobic transport device or a sterile tube or
petri dish. All this is placed into a sealable plastic bag that
generates an anaerobic atmosphere.
 If specimens must be collected by swab, swabs should be
transported in a tube containing anaerobic transport medium.
 Transport containers –Syringe or needle for aspiration, Tube
or vial,
Swab/plastic jacket system, Bio-bag or plastic pouch.

59. Fungal Culture
• Respiratory specimens- Sputum, tracheal secretions and BAL
fluid.
• CSF
• Blood
• Tissue, Bone Marrow and Body Fluids
• Skin, Hair and Nail-
 Skin scraping.
 Nail clippings should be taken from discoloured, dystrophic or
brittle parts of nails.
 The hairs are removed by plucking them with forceps
• Urine
• Vaginal secretions
• Stool
• Eye-
 corneal scrapings are taken from base and margins of ulcer
aseptically using Kimura’s spatula under 4% xylocaine

60. Collection of Faecal samples for Parasitology
• All faecal specimens should be collected prior to the
administration
of antibiotics or antidiarrhoeal agents.
• Use of mineral oil, bismuth, and barium prior to faecal collection
should be avoided
• Faecal specimen is collected in a clean, wide mouthed container
and transfer it to a container with a tight-fitted lid.
• Specimen transport
 Specimen is transported to the lab as soon as possible, or kept
refrigerated until transport is possible.
• Rejection criteria
 Dried specimens
(diarrhoeic, semiformed, or formed.) Stool sample
container

61. ANALYTICAL PHASE
Blood Culture
•Macroscopic signs of
growth caused by
organisms commonly
encountered in blood
culture
Haemolysis-
Streptococci,
Staphylococci, Listeria
spp, Clostridia ,Bacillus
Turbidity- Aerobic GNB,
Staphylococci,
Bacteroides.
 Gas formation- Aerobic GNB, anaerobes
 Pellicle formation- Pseudomonas spp., Bacillus spp., yeast cells
 Clotting- Staphylococcus aureus
 Visible colonies (“puffballs”)- staphylococcus, streptococci
• Microscopy -
raga ro htorb eht morf deniats marG si raems niht A
immediately when suggestive of growth 24h for optimal pt. care.
• Subcultured to agar media and biochemical tests based on
Gram stain results.
Bac T
Alert

62. Lower Respiratory Tract Cultures
• Inoculation
 Most purulent or most blood–tinged portion of the specimen is
selected
 In case of bronchoscopy, quantitative cultures are performed on
BAL samples
 Gram stain is prepared
 Using a sterile swab, stick or pipette,
inoculate specimen on blood agar, chocolate agar, and
MacConckey agar
• Incubation
 Plates are incubated at 35 to 370C in 5% CO2 for a minimum of
48 h.

63. • Direct Tests
 Gram stain: note cells and bacteria
• Culture examination
 Plates are observed at 24 h
 Plates are incubated for an additional 24 to 48 h, which is
useful to
detect moulds and slow growing, fastidious gram negative rods
 Gram stain result is used as a guide to interpreting the
culture.
 Presence of inflammatory cells and bacteria is used in
deciding on the extent of processing the culture
If the culture does not match the smear results, the smear
is
reviewed a second time.

64.  Organisms present in significant amounts are identified,
defined as colony types that are not considered part of
normal respiratory microbiota and are present in following
amounts:
Large numbers in the second or greater quadrant of the
plate
 >
104 in a BAL sample
Any amount of selected pathogens in a patient with cystic
fibrosis
 Small amount of selected pathogens in the culture that
are consistent with an etiological agent seen in the
Gram stain associated with inflammatory cells
 Colonies in the first quadrant of the plate, only if there is
little or no other microbiota on the plate and the smear
suggests inflammation
 Subcultured on blood agar &/or chocolate agar to obtain
isolated colonies for accurate identifications from mixed
cultures
• Biochemical tests and AST

65. Urine Cultures
• Microscopic and other methods
 Gram stain is useful in rapidly determining the type and count of
bacteria and cells in urine and should be performed on request.
 Detection of pyuria- Detect by either Gram stain or urinalysis,
from examination of freshly collected uncentrifuged urine.
Pyuria with a WBC count of > 10
> ro( lµ/
5 per high-power field
in conventional urinalysis) has a specificity of 90% for
predicting CA-UTI with >105 CFU/ml but a sensitivity of only
37%
.
• Culture methods
 Only streak the blood plate for colony count. Other plates should
be streaked in quadrants for isolation of colonies, rather than
for colony count, to minimize delays in obtaining isolated
colonies and false-negative culture results due to antimicrobial
inhibition.
 Inoculation methods: Calibrated-loop method, Pipette
inoculation

66. • Incubation
 In ambient air overnight at 35 to 370C
 If convenient, blood agar and CNA is incubated in 5% CO2 to
enhance growth of gram- positive organisms.
• Examination of culture media
 Cultures that have been incubated overnight are examined but a
final reading is made at 18 h.
 For positive cultures, culture media is examined for quantity and
morphological type of organisms present
 Normal urogenital flora is not identified to the genus or species
level
 Identification is done always to the species level if oxidase
positive and indole positive, since such organisms are
pathogens regardless of the count (eg. Aeromonas and Vibrio
spp.)

67. Wound and Soft tissue culture
• Inoculation
 Anaerobic culture is performed first, preferably in an
anaerobic chamber. Incubated immediately.
 Smear is always prepared after the culture has been
inoculated.
• Gram stain
 Relative numbers of WBCs, epithelial cells, and bacterial
and fungal morphotypes are recorded
• Culture workup
 Plates and broths are read daily

68.  Generally up to three microorganisms are identified if any of
the following is true:
 PMNs are present on direct smear
 Specimen was collected from a normally sterile site
 The specimen was of good quality (eg. No or few
epithelial cells present)
 The organism was seen on direct smear
 Any number of microorganisms that only grow on chocolate
agar, and not on blood agar ( N. gonorrhoea, Haemophilus,
and Franciselladeifitnedi era )
• Microorganism is identified and AST is performed

69. Faecal Culture for
Aerobic Pathogens
• Wet preparations are performed for faecal leukocytes from
fresh stools.
• Culture Methods
 Inoculation of media- Swab is rolled over one small area of
blood agar and Mac Conckey media and streaked in
quadrants for isolated colonies. Use larger amounts of
specimen for HEK, XLD and SS agars.
• Biochemical tests performed
• Salmonella and Shigella somatic (O) antigens are tested for
agglutination when the screening biochemical tests fit.

70. Anaerobic Culture
• Macroscopic Examination
 Presence of blood, purulence, necrotic tissue, foul odour, and
sulphur granules.
• Specimen preparation
 Purulent specimens are vortexed grossly in the anaerobic
transport vial to ensure even distribution of microorganisms.
 Bone or tissue are grounded with approximately 1ml of liquid
medium to make a thick paste.
 Swabs are wringed out in 0.5ml of liquid medium and then
treated as a liquid specimen
 Large volume of nonpurulent material is centrifuged.
Sediment is
used to inoculate the media and to prepare the Gram stain
• Inoculation of media
 Media for anaerobic culture:

71. Brucella agar with 5% sheep blood supplemented with
vitamin K and hemin for isolation of most organisms
Phenylethyl alcohol (PEA)- sheep blood agar for the
inhibition of enteric and other facultatively anaerobic
gram-negative bacilli that may overgrow the anaerobes.
PEA also reduces the spreading or swarming
characteristic of some anaerobes.
Kanamycin-Vancomycin-laked blood agar for selection of
pigmented Prevotella and other Bacteroides spp.
Bacteroides bile esculin agar
Chopped meat broth or THIO (supplemented with Vit K&
hemin)
Prereduced anaerobically sterilized (PRAS) media

72. • Inoculation procedure
 Prepared specimen is transferred onto the appropriate aerobic
and
anaerobic media, liquid medium and a slide for Gram stain.
• Incubation
• Microscopic Examination
 Gram staining: Reveals the types
and relative numbers of microorganisms and host cells present
and serves as a
QC measure for the adequacy of anaerobic echniques.
Anaerobic Chamber

73. Fungal
Culture
• Inoculation of media
 The number of media inoculated may be dependent on the
specimen type and amount.
 Both non-selective (SDA and BHI) and selective media
should be included, and specialized media may be required
for specimens in which difficult-to-grow etiological agents
are suspected.
• Incubation
 Fungal cultures are incubated at 300C for
a minimum of 4weeks.
 Plates should be examined daily for first
7days and at least twice per week thereafter.

74. Faecal sample examination for Parasites
• Macroscopic Examination
 Consistency
 Surface of faecal specimen for the presence of blood and
mucus.
 Areas of blood or mucus are sampled for examination of
trophic amoeba
• Microscopic examination
 Saline and iodine wet mounts are prepared
 Examined under 10x. Use 40x
objective for more detailed study.
• Floatation and concentration methods
are used for the recovery of all protozoa,
eggs, and larvae present. Iodine and
Saline
mounts

75. Post-analytical Phase
Blood culture
•Reporting results
For “No growth cultures ”
:detacidni si noitabucni fo htgnel ,
“No growth after x days of incubation ”for both preliminary and
final reports.
Positive cultures:
Gram stain results of all positive cultures are reported
immediately
Number of positive cultures compared with total number
of specimens collected for specific patient.
Date and time of collection and receipt.
Date and time of positive result is reported and whether it
was from a catheter draw or a peripheral draw.

76. For single positive cultures with microorganisms generally
considered skin contaminants, only minimal identification is
performed and AST is not done. Reported as “one set of two
positive. Isolation does not necessarily mean infection. No
susceptibility tests performed. Contact laboratory for further
information .
”
•Interpretation
The report of a positive culture generally means that the patient is
bacteremic. However, skin microbiota may infect the culture, causing
a false-positive result or pseudobacteremia.
Mixed cultures can be present and account for a significant number
of bacteremias.
Performance and reporting of AST are critical for timely patient care
and increase the chance of appropriate therapy and cure.

77. Lower Respiratory Tract Cultures
• Reporting results
 Gram stained smear is reported. Rare (<1 in 20 fields) numbers of
bacteria seen in the smear are not reported.
 Reported as: “No growth of pathogens or normal upper respiratory
tract microorganisms” if there is no growth on any plates
 Positive reporting
Preliminary and final results are reported as “Isolates consistent
with microorganisms encountered in the upper respiratory
tract” if no pathogens isolated
All pathogens are reported and AST is performed.

78. • Interpretation
 A positive culture with S. pneumoniae or H. influenzae
generally indicates infection with that organism,
although carriage may lead to false positive results.
 A positive culture with a predominant gram-negative
rod or
S. aureus generally indicates infection with that organism
if smear suggests an infectious process involving the
corresponding morphology.
 A negative culture cannot rule out infection.

79. Urine Culture
• Reporting results
 Gram stain results for bacteria & cells per Gram stain are
reported.
 Negative results
If no growth is observed on all media, report “No
growth after 48hrs“
 Positive results
If only urogenital or skin microbiota is observed, report
as such.
Mixed cultures are reported with the count in CFU per
millilitre, followed by “Multiple bacterial morphotypes
present; possible contamination; suggest appropriate
recollection, with timely delivery to the laboratory, if
clinically indicated”

80. • Interpretation
 A mixed culture in an uncomplicated outpatient population
likely indicates contamination.
 Low levels (< 104
eht no dnuof ylnommoc smsinagro fo )lm/
ot deredisnoc era ailatineg lanretni dna lanretxe dna niks
fo tnuoc a ,secnatsmucric detceles ni tub ,stnanimatnoc eb
fo eacairetcaboretnE
102 CFU/ml or more, especially for
Salmonella tnacifingis deredisnoc eb nac ,
.
 Performance of AST directly from the urine specimen is not
recommended.

81. Wound and soft tissue infections
• Reporting results
 Report gram stain results as soon as possible, generally within 1
h for specimens from critical sites.
 Report all negative cultures as “no growth in-------days”
 Report individually those organisms that are always considered
pathogenic with enumeration, using preliminary identification
initially and the genus and species as the final identification.
 Due to their known virulence factors, indicate the presence of the
following species: Beta-haemolytic Streptococci, S. aureus, P.
aeruginosa, Clostridium perfringens ,seboreana detnemgiP ,
Bacteroides spp., Mixed anaerobes.
 Report AST on gram-negative rods, enterococci, or S. aureus
 When multiple morphologies are present, report with minimal
identification
 Additionally, if mixed microbiota are cultured with no predominant
microorganism, report as GI, oronasal, skin, or genital microbiota.

82. • Interpretation
 Performance of AST is not indicated in cases of mixed
microbiota indicative of infection of the abdominal cavity with
bowel contents. Treatment should include broad-spectrum
coverage for normal intestinal microbiota.
 Use of the Gram stain can improve the accuracy of evaluating
the importance of each potential pathogen. Organism
present in the Gram stain of an appropriately collected
specimen correlate with
≥
105 organisms per g of tissue.
 Microbial load in an acute wound can predict delayed healing
or infection.
 Many wound infections are polymicrobic, and the isolation of
an organism in culture may or may not correlate with
infection of the wound.

83. Faecal culture for aerobic pathogens
• Reporting results
 If gram negative enteric microbiota are not present in the cultures,
add a comment “no normal enteric gram-negative rods isolated ”
.
 Positive cultures: report presumptive presence of any enteric
pathogens with or without quantitation. Report the pathogen with
the preliminary designation as “probable ”until the identification is
confirmed by both biochemical testing and serology.
 Report AST on Shigella, Aeromonas, Plesiomonas, Edwardsiella,
Vibrio, Yersinia and selected cases of Salmonella.
 Generally report only ampicillin, cotrimoxazole, and a quinolone.
• Interpretation
 The isolation of a stool pathogen may not identify the cause of the
disease. Eg. Salmonella is present in the carrier state, without
disease.

84. Fungal culture
• Reporting and interpretation of yeast:
Nitrate assimilation test for yeast speciation
Sporulation
Carbohydrate fermentation tests
• Reporting and interpretation of mould:
Reporting the growth of a mould as soon as such growth is
evident but before identification structures are formed is
important where immunocompromised patients are concerned
but is not normally necessary in cases of subcutaneous
infection of immunocompetent patients.

85. Parasitic infestations
• To ensure the recovery of parasitic organisms that are
passed intermittently and in fluctuating numbers, the
examination of a minimum of three specimens collected
over a 7
-
ot
10
-
dednemmocer si doirep yad
.
• Report the presence of adult helminths .Morphology and
size are usually adequate for identification of pinworm and
Ascaris adults and tapeworm proglottids.
• Report the presence of blood on or in the faecal sample

86. COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Collecting Blood
Clean with 70% ethyl alcohol
Disinfect with 10% povidone-iodine
Allow to dry for at least 1 minute
No wiping!
Clean the rubber stopper of the bottle
Use alcohol for Bactec bottle to prevent cracking

87. COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Collect enough blood
1-2ml in neonate
2-3ml in infants
3-5ml in children
10-20ml in adolescent
Rapid inoculation
A 3 hour delay result in 25% reduction in recovery of S.
pneumoniae
Paisley JW, Lauer BA. Pediatric blood cultures. Clin Lab Med 1994; 14: 17
Roback MG, Tsai AK, Hanson KL. Delayed incubation of blood culture bottles: Effect
on recovery rate of S. pneumoniae. Pediatr Emerg Care 1994; 10: 268

88. COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Collecting urine
Clean-voided midstream urine
Use of urine bag
Catheterized specimen/ Suprapubic aspiration
Collecting CSF
CSF is hypotonic
Refrigeration can render fastidious bacteria non-viable
Cell count decreases by 32% after 1 hour and 50% after 2 hours
Steele RW, Mormer DJ, O’Brien MD, et al. Leukocyte survival in
cerebrospinal fluid. J Clin Microbiol 1986; 23: 965

89. COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Insufficient quantity/quality
Small quantity for optimal analysis
Poor specimen e.g. eye cultures for chlamydiae should
have enough cellular element
Contamination
During collection
During transport
Contamination in the lab

90. COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Improper transport media
Prevent drying
Maintain optimal physiochemical environment
Prevent oxidation and destruction of enzymes
Provide adequate nutrients
Three major culture media
Enrichment: chocolate and sheep blood
Selective: Thayer-Martin
Differential: MacConkey-ability to ferment lactose

91. COLLECTION AND PROCESSING OF
CLINICAL SPECIMEN
Delay in transportation
Holding conditions are specimen or pathogen
specific
Urine: 2˚ C to 8˚C
Inoculated blood: 35˚ C to 37˚C

92. SPECIFIC EXAMPLES
Specimen for isolation of N. gonorrhoeae should be
inoculated into a specific media, transported within 30
minutes of collection, incubated at 35˚-37˚C in 5-10% co2
Stool for ova & parasite should be placed in preservatives
CSF is held in room temperature and never refrigerated
Stool for C. difficle must be refrigerated or frozen

94. Laboratory Investigation of Microbial infections
Examining specimens to detect isolate and identify pathogens:
1- Microscopy
2- Culture techniques
3- Biochemical reactions
4- Serological identification:
5- Molecular biology techniques
6- Bacteriophage typing

95. 1- Microscopy
Microorganisms can be examined microscopically for:
a- Bacterial motility:
Hanging drop method:
A drop of bacterial suspension is placed between a cover slip and glass slid
b- Morphology and staining reactions of bacteria:
Simple stain: methylene blue stain
Gram stain: differentiation between Gm+ve and Gm–ve bacteria
. Primary stain (Crystal violet)
. Mordant (Grams Iodine mixture)
. Decolorization (ethyl alcohol)
. Secondary stain ( Saffranin)
Ziehl-Neelsen stain: staining acid fast bacilli
. Apply strong carbol fuchsin with heat
. Decolorization (H2SO4 20% and ethyl alcohol
. Counter stain (methylen blue)

96. 2- Culture Techniques
* Culture media are used for:
- Isolation and identification of pathogenic organisms
- Antimicrobial sensitivity tests
* Types of culture media:
a- Liquid media:
- Nutrient broth: meat extract and peptone
- Peptone water for preparation sugar media
- Growth of bacteria detected by turbidity
b- Solid media:
- Colonial appearance
- Hemolytic activity
- Pigment production

97. Types of solid media
1- simple media:
Nutrient agar
2- Enriched media: media of high nutritive value
. Blood agar
. Chocolate agar
. Loffler’s serum
3- Selective media: allow needed bacteria to grow
. Lowenstein–Jensen medium
. MacConkeys agar
. Mannitol Salt Agar
4- Indicator media: to different. between lact. and non lact. ferment
. MacConkeys medium
. Eosine Methlyne blue Agar
5- Anaerobic media: for anaerobic cultivation
. Deep agar, Robertson’s Cooked Meat Medium

98. Colonial appearance on culture media
* Colony morphology:
. Shape . Size . Edge of colony . Color
* Growth pattern in broth:
. Uniform turbidity
. Sediment or surface pellicle
* Pigment production:
. Endopigment production (Staph. aureus)
. Exopigment production (Ps. aeruginosa)
* Haemolysis on blood agar:
. Complete haemolysis (Strept. Pyogenes)
. Partial haemolysis (Strept. Viridans)
* Growth on MacConkey’s medium:
. Rose pink colonies (Lactose fermenters)
. Pale yellow colonies (Non lactose fermenters)

99. 3- Biochemical Reaction
Use of substrates and sugars to identify pathogens:
a- Sugar fermentation:
Organisms ferment sugar with production of acid only
Organisms ferment sugar with production of acid and gas
Organisms do not ferment sugar
b- Production of indole:
Depends on production of indole from amino acid tryptophan
Indole is detected by addition of Kovac’s reagent
Appearance of red ring on the surface
e- H2S production:
Depends on production H2S from protein or polypeptides
Detection by using a strip of filter paper containing lead acetate

100. 3- Biochemical Reaction (cont.)
c- Methyl red reaction (MR):
Fermentation of glucose with production of huge amount of acid
Lowering pH is detected by methyl red indicator
d- Voges proskaur’s reaction (VP):
Production of acetyl methyl carbinol from glucose fermentation
Acetyl methyl carbinol is detected by addition KOH
Color of medium turns pink (positive)
e- Action on milk:
Fermentation of lactose with acid production
Red color if litmus indicator is added

101. 3- Biochemical Reaction (cont.)
f- Oxidase test:
Some bacteria produce Oxidase enzyme
Detection by adding few drops of colorless oxidase reagent
Colonies turn deep purple in color (positive)
g- Catalase test:
Some bacteria produce catalase enzyme
Addition of H2O2 lead to production of gas bubbles (O2 production)
h- Coagulase test:
Some bacteria produce coagulase enzyme
Coagulase enzyme converts fibrinogen to fibrin (plasma clot)
Detected by slide or test tube method
i- Urease test:
Some bacteria produce urease enzyme
Urease enzyme hydrolyze urea with production of NH3
Alklinity of mediaand change color of indicator from yellow to pink

102. 4- Animal pathogenicity
* Animal pathogenicity test:
Animals commonly used are guinea pigs, rabbits, mice
* Importance of pathogenicity test:
- Differentiate pathogenic and non pathogenic
- Isolation organism in pure form
- To test ability of toxin production
- Evaluation of vaccines and antibiotics

103. Serological identification
A- Direct serological tests:
- Identification of unknown organism
- Detection of microbial antigens by using specific
known antibodies
- Serogrouping and serotyping of isolated organism
B- Indirect serological tests:
- Detection of specific and non specific antibodies
(IgM & IgG) by using antigens or organisms

104. DIAGNOSTIC TECHNOLOGIES
Antigen Detection Assays
Most has poor sensitivity and specificity
57% sensitivity and 98% specificity for pneumococcal pneumonia
Conc. Urine EIA for Legionella pneumophila serogroup 1 has 89%
sensitivity and 100% specificity
Immunochromatographic assay has better
sensitivity and are faster

105. DIAGNOSTIC TECHNOLOGIES
Immunoserology
Hemagglutination
EIA
Latex agglutination
Complement fixation
Immunoflorecent

106. Serologicalmethods:
There are a variety of serological assays available
including
Several of these tests can measure antibody titer
by performing dilutions of patient serum to
determine the lowest titer at which reactivity is
seen.
1-ELISA.

107. RAPID DIAGNOSTIC TESTS
High sensitivity and specificity
High negative and positive predictive values
High accuracy compared to gold standard
Simple to perform
Rapid turn around time
Cost effective

108. Pathogens Generic name of test Mechanism Sensitivity
(%)
Specificity
(%)
Positive
Predictive
Value (%)
Negative
Predictive
Value (%)
Time to perform test
Respiratory Syncytial Virus RSV Immunoassay Qualitative detection of RSV
antigen by
immunoassay
89-93 93 - 99 96-97 80-89 15 minutes
Influenza A Virus Rapid Flu A Detection of influenza A
nucleoprotein
antigen
78-82 92 - 94 80-91 90-97 10-15 minutes
Influenzas B Virus Rapid Flu B Detection of influenza B
nucleoprotein
antigen
58-71 96 - 97 Not available Not available 10-15 minutes
Influenza A & B Viruses Rapid Flu A & B Non-differential detection of
both influenza A &
B by
neuraminidase
enzyme assay
52-73 92-99 95-98 74-80 22 minutes
Influenza A + B Viruses Influenza A + B Differential detection of both
influenza A & B by
Immunoassay
72 – 82 96 – 99 80-90 90-97 10 minutes
Epstein Barr Virus Mono Spot Detection of heterophile
antibodies
91-99 96 97-98 99 3-5 minutes
HIV Rapid HIV Detects HIV-1 antibody 99 99 Not available Not available 10-20 minutes

109. Pathogens Mechanism Sensitivity
(%)
Specificity
(%)
Positive
Predictive
Value (%)
Negative
Predictive
Value (%)
Time to perform
test
Group A
Streptococcus
Detects group A
staphylococcal
carbohydrate
antigen by
immunoassay
89-94
(Compared to
culture)
95-99 55-89 90-97 5 minutes
Helicobacter
pylori
Detects
immunoglobulin
G antibodies
specific to H.
pylori
85-90
(Compared to
biopsy)
80-89 85 79 5-10 minutes
Borrelia
burgdorferi
Detects
antibodies to B.
burgdorferi using
recombinant
antigen
72
(Compared to
ELISA)
97 Not available Not available 20 minutes

110. LIMITATIONS OF CONVENTIONAL
CLINICAL MICROBIOLOGY
Culture
Labor intensive
Need for special media
Prolonged period of time to culture
Some organisms are uncultivable on artificial media
Potential health hazards
Antigen Detection
Negative tests require confirmation
Effected by poor specimen collection
Low microbe burden
Serology
Unhelpful during early stage of infection
Not quite useful in immunocompromised patients

111. Molecular Biology Techniques
A- Genetic probes (DNA or RNA probes):
Detection of a segment of DNA sequence (gene) in unknown
organism using a labeled probe
Probe: consists of specific short sequence of labeled single-
stranded DNA or RNA that form strong covalently
bonded hybrid with specific complementary strand of
nucleic acid of organism in question
B- Polymerase chain reaction (PCR):
Amplification of a short sequence of target DNA or RNA Then
It is detected by a labeled probe
C- Plasmid profile analysis:
Isolation of plasmids from bacteria and determination of their
size and number compared with standard strains by agarose
gel electrophoresis

112. MOLECULAR DIAGNOSTICS
Most widely used is PCR
High sensitivity
High specificity
Diversity
Nucleic acid probes
Do not amplify DNA

113. MOLECULAR DIAGNOSTICS
Polymerase Chain Reaction
Specific PCR: Uses primers to known DNA targets. So
far 31 clinical bacterial gene sequence are known and
38 in progress
• Use when conventional diagnostics are inadequate, time
consuming, difficult and hazardous
Broad range PCR: uses complementary primers to
conserved regions shared by a given taxonomic group
• Used in cases of B. henselae and Mycobacterium spp

114. MOLECULAR DIAGNOSTICS
Multiplex PCR
Uses single clinical specimen to investigate several
potential pathogens simultaneously
• Encephalitis/meningitis panel: HSV,VZV, CMV HHV-6,
EBV, Enteroviruses
Real-time PCR
Utilizes a fluorescent labeled probe
Requires small volumes thus takes 30-60 minutes to
complete

115. OTHER USES OF MOLECULAR
DIAGNOSTICS
Viral load monitoring
Viral genotyping
Bacterial resistance detection
Bacterial genotyping

116. B-PCR
AmplificationsegmentofDNAorRNAmanytimesto
detectit.

117. C. Bacterial Identification Using 16S rRNA Probe Hybridization
The 16S rRNA of each species of bacteria has stable (conserved) portions of
the sequence. Many copies are present in each organism. Labeled probes
specific for the 16S rRNA of a species are added, and the amount of label on
the double stranded hybrid is measured.
EX: Histoplasma capsulatum

118. LIMITATION OF PCR TECHNOLOGIES
Specimen should be frozen until
amplification
No antimicrobial sensitivity is available
Needs the clinician to name the suspect

119. LIMITATION OF PCR TECHNOLOGIES
Cost
False positives caused by amplification of
contaminants
Only sample from normally sterile sites should be
considered for broad-range PCR
Specimen is required to be refrigerated or stored in
alcohol before processing

120. Practical applications using phages
* Phages are important as a research tools
* Phages are used as vectors in DNA recombinant
technology
* Phage typing of bacteria is important in tracing
source of infection for epidemiologic purposes

121. 7-Bacteriophages
Bacteriophage (phage) are obligate
intracellular parasites that multiply inside
bacteria by making use of some or all of the
host biosynthetic machinery.
1-generalizedtransduction
2-specializedtransduction

122. 1-generalized transduction: A DNA fragment is
transferred from one bacterium to another by a lytic
bacteriophage that is now carrying donor bacterial
DNA due to an error in maturation during the lytic
life cycle.

123. 2-specialized transduction:A DNA fragment is transferred
from one bacterium to another by a temperate bacteriophage that is now carrying
donor bacterial DNA due to an error in spontaneous induction during the
lysogenic life cycle

124. 8-Animal pathogenicity
use of non-human animals in experiments that seek to control
the variables that affect the behavior or biological system
under study.
Animals commonly used : guinea pigs,
rabbits, mice
Importance of pathogenicity test: -
1-Differentiate pathogenic and non
pathogenic
2- Isolation organism in pure form
3- To test ability of toxin production
4- Evaluation of vaccines and antibiotics

125. Antimicrobial Susceptibility testing
Introduction:
Identification of a bacterial isolate from a patient provides
guidance in the choice of an appropriate antibiotic for
treatment
Many bacterial species are not uniformly susceptible to a
particular anti-bacterial compound
This is particularly evident among the Enterobacteriaceae,
Staphylococcus spp., and Pseudomonas spp.
The wide variation in susceptibility and high frequencies of
drug resistance among strains in many bacterial species
necessitates the determination of levels of resistance or
susceptibility as a basis for the selection of the proper
antibiotic for chemotherapy

126. Antimicrobial Susceptibility testing can be down
by three ways:
1. Minimum Inhibitory Concentration (MIC)
2. Disk Diffusion Method
3. Minimum Bactericidal Concentration (MBC)

127. 1. Minimum Inhibitory Concentration (MIC) :
Principle:
The tube dilution test is the standard method for
determining levels of resistance to an antibiotic.
Serial dilutions of the antibiotic are made in a liquid
medium which is inoculated with a standardized number
of organisms and incubated for a prescribed time.
The lowest concentration of antibiotic preventing
appearance of turbidity is considered to be the minimal
inhibitory concentration (MIC).

128. Different concentrations of Gentamycin in Nutrient broth:
Conc. in mcg/ml
0.1 0.2 0.4 0.8 1.6 3.1
Gentamicin, generally considered a bacteriocidal antibiotic, for this
bacterium, has an MIC of 0.8 mcg/ml

129. Different concentrations of Tetracycline in Nutrient broth:
Conc. in mcg/ml
0.1 0.2 0.4 0.8 1.6 3.1 6.3 12.5
Tetracycline, generally considered a bacteriostatic antibiotic, for this
bacterium, has an MIC of 1.6 mcg/ml

130. 2. Disk-diffusion Method (Kirby-Bauer Method):
The disk-diffusion method (Kirby-Bauer) is more suitable for
routine testing in a clinical laboratory where a large number of
isolates are tested for susceptibility to numerous antibiotics.
An agar plate is uniformly inoculated with the test organism
A paper disk impregnated with a fixed concentration of an
antibiotic is placed on the agar surface.
Growth of the organism and diffusion of the antibiotic commence
simultaneously resulting in a circular zone of inhibition in which
the amount of antibiotic exceeds inhibitory concentrations.
The diameter of the inhibition zone is a function of the amount of
drug in the disk and susceptibility of the microorganism.

131. This test must be rigorously standardized since zone size is also
dependent on:
• inoculum size,
• medium composition,
• temperature of incubation,
• excess moisture and
• thickness of the agar.
Zone diameter can be correlated with susceptibility as
measured by the dilution method.
Further correlations using zone diameter allow the designation
of an organism as "susceptible", "intermediate", or "resistant"
to concentrations of an antibiotic which can be attained in the
blood or other body fluids of patients requiring chemotherapy.

132. Using a dispenser, antibiotic-impregnated disks are placed onto the agar
surface.
As the bacteria on the lawn grow, they are inhibited to varying degrees by
the antibiotic diffusing from the disk.

133. Staphylococcus aureus (MRSA)
Note the yellowish pigmentation of the bacterial lawn, and the
lack of inhibition by the Oxacillin disk

134. Streptococcus pneumoniae (Pneumococcus):
The brownish tint of the blood agar plate outside the zones of
bacterial inhibition is caused by alpha-haemolysis.

135. Pseudomonas aeruginosa:
The greenish tint of the lawn and plate in general is caused by the
diffusible pigment made by the Pseudomonas aeruginosa itself.

136. Examples :
case study examination

137. You are asked to attend the delivery of a term infant because the baby is small
for gestational age, and prenatal ultrasonography revealed periventricular
cerebral calcifications. The infant’s birth weight is 2,000 g. On physical
examination, you note hepatosplenomegaly and a petechial rash on the face and
trunk.
Of the following, the BEST laboratory test for diagnosing the cause of these findings is
A. Nasopharyngeal culture for herpes simplex
B. Rapid plasma reagin for syphilis
C. Serum immunoglobulin (Ig) G titer for rubella
D. Serum IgM titer for toxoplasmosis
E. Urine culture for cytomegalovirus

138. A 12-year-old girl comes to your office complaining of headache, malaise, fever to
101 F (38.3  C), rhinorrhea, and a sore throat for the past 3 to 4 days. Most recently,
she developed hoarseness and cough. On physical examination, you note crackles and
wheezes throughout the lung fields. Chest radiography demonstrates interstitial
infiltrates in the lower lung fields bilaterally.
Of the following, the BEST test to order to confirm the patient’s diagnosis
is:
A. Direct fluorescent antibody for Bordetella pertussis
B. Enzyme immunoassay for respiratory syncytial virus
C.Serology for Mycoplasma pneumoniae
D.Throat culture for group A Streptococcus
E. Viral culture for parainfluenza

139. A 16-year-old boy presents with a 5-day history of low-grade fever, headache,
mild nasal congestion, and a persistent cough associated with posttussive
syncope, he reports that it is hard for him to catch his breath after one of his
coughing episodes. His immunizations are up to date. Several of his classmates
are ill with similar symptoms. Chest radiography results are normal.
Of the following, the test that is MOST likely to aid in the diagnosis of
this patient is:
A. Cold agglutinin test
B. Mantoux skin test with purified protein derivative
C.Monospot
D.Pertussis direct fluorescent antibody
E. Sputum Gram stain

140. A 5-year-old girl develops fever, swelling of the parotid
gland, and headache.
Of the following, the BEST diagnostic test for this child is:
A. Bacterial culture of parotid duct secretions
B. Epstein-Barr virus serology
C.Mumps serology
D.Serum amylase
E. Viral culture of respiratory secretions

141. A 3-year-old boy presents with a large, non-tender, rubbery anterior
cervical lymph node. You prescribe a course of dicloxacillin, but there is
no change in the node. Results of Mantoux purified protein derivative
skin test reveal 8 mm in duration.
Of the following, the BEST diagnostic procedure to undertake in this
patient now is:
A. Biopsy of the node
B. Chest radiography
C.Excision of the node
D.Gastric aspirates for culture
E. Needle aspiration of the node

142. A newborn has evidence of symmetric intrauterine growth restriction.
Evaluation reveals microcephaly with intracranial calcifications, “salt and
pepper” retinopathy, hearing deficit, enlarged liver and spleen, and
purpura. Laboratory evaluation documents thrombocytopenia.
Of the following, the test MOST likely to confirm the diagnosis in this
infant is:
A. Cytology of a conjunctival swab
B. Rapid plasma reagin test
C. Serology of blood
D. Urine assay for interferon
E. Viral culture of urine

143. Of the following, the BEST direct stain to detect
Mycobacterium tuberculosis is the
A. Calcofluor white stain
B. Giemsa stain
C. Gram stain
D. Kinyoun stain
E. Periodic acid-Schiff stain

144. A 9-year-old boy has had a nonproductive cough for the past 3 weeks. He has
been afebrile and otherwise feeling well. On physical examination, you note
widespread rales. Chest radiography reveals bilateral, diffuse infiltrates. You
diagnose pneumonia, most likely due to Mycoplasma pneumoniae.
Of the following, the test that would BEST confirm the diagnosis is:
A. Bacterial culture of sputum
B. Blood culture
C.Gram stain of sputum
D.Mycoplasma-specific immunoglobulin M
E. Serum cold agglutinins

145. A 5-year-old boy is hospitalized in January with fever and seizures.
Lumbar puncture reveals clear cerebrospinal fluid that has a white
blood cell count of 47/ cu mm, all of which are lymphocytes. On
physical examination, he appears obtunded but arouses with painful
stimuli. Neurologic examination reveals no focal findings.
Of the following, the diagnostic test that is MOST likely to reveal the
etiology of this child’s illness is:
A. Bacterial culture of cerebrospinal fluid of herpes simplex
B. Polymerase chain reaction test of cerebrospinal fluid for
herpes simplex
C. Streptococcus pneumoniae bacterial antigen test of
cerebrospinal fluid
D. Viral culture of cerebrospinal fluid
E. Viral culture of nasopharyngeal and rectal swabs

146. A newborn has hepatosplenomegaly, purpuric rash, jaundice,
thrombocytopenia, and microcephaly. Computed tomography
of the head demonstrates cerebral calcifications
Of the following, the MOST appropriate diagnostic testing for this infant
includes:
A. Maternal human immunodeficiency virus serology
B. Serologic testing of mother and infant for
cytomegalovirus
C.Serologic testing of mother and infant for Toxoplasma
D.VDRL on infant and maternal sera
E. Viral culture of swabs of infant’s throat and
conjunctivae

147. A 12-year-old girl is brought to your office of evaluation
following 4 weeks of diarrhea, abdominal pain, and weight
loss. You suspect giardiasis.
Of the following, the MOST reliable next step to establish the diagnosis
is to examine a single stool sample for:
A. Giardia antigen
B. Leukocytes
C.Ova and parasites
D.pH
E. Reducing substances

148. A 4-month-old infant develops severe paroxysmal coughing
10 days after the onset of nasal congestion and rhinorrhea.
His mother reports that often 15 to 20 coughs occur in rapid
succession.
Of the following, the BEST test to establish the diagnosis is:
A. Bronchoscopy that demonstrates the presence of a
foreign body
B. Culture of a nasal swab that grows a small gram-
negative coccobacillus
C.Culture of a nasal swab that shows viral growth
D.pH probe that demonstrates gastroesophageal reflux
E. Skin testing with demonstration of allergies

149. Thank you
Principle Laboratory Diagnosis of Infectious Diseases