MICR 301 – Fall, 2011
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Office Hours
Lecture Schedule & Reading
Texts
Case Study Reports
Websites
Course Prerequisite
Course Objectives
Class Attendance
Student Evaluation & Grading
Academic Honesty
Model of Creative Problem Solving
Critical Thinking Skills
Laboratory Schedule
QUESTIONS???
General Medical
Microbiology
Specimen Collection and Processing
Specimen Collection
• Failure to isolate causative agent of
infectious disease frequently result of
faulty collecting or transport techniques
• Therefore, when collecting specimen for
microbiological examination, several
general considerations need to be
addressed
Specimen Considerations
• Representative of disease process
– i.e. for pneumonia - sputum not throat swab
• Sufficient material collected
• Avoid contamination by patient’s microbial
normal flora
• Collect before antibiotic therapy started
• At acute phase of disease
• Delivered promptly to lab
• Clinical information to guide culture and ID
Sterile Specimen
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Blood
Cerebral spinal fluid (CSF)
Tissue
Serous fluids
Specimens from the lower
respiratory tract (LRT)
• Urine directly from bladder or kidney
Specimen With Microbial
Normal Flora (NF)
• Upper respiratory tract (URT), including
mouth and nose
• Sputum (LRT)
• Feces
• Genital tract
• Skin
• Low number organisms:
– Conjunctiva of eye
– External ear
Circumventing Normal
Flora
• Antiseptics (iodine, alcohol) - apply to skin
prior to aspiration of abscess, blood, CSF
• Decontamination – selectively inhibit or
destroy NF i.e. treat sputum with NaOH
before culturing for Mycobacteria
• Selective media – inhibit growth of NF and
allow growth of pathogen i.e. bile salts for
Enterics
• Quantification – >#MOs than expected;
i.e. procedure used for urine culture
Urine Culture
• A calibrated loop delivers 1 ul of
urine (10-3ml)
• If colony count >100 colonies (#MOs
>105/ml) is considered significant and
indicates infection
Circumventing Normal
Flora - Microscopy
• Cytological exam - look for presence of
squamous epithelial cells in urine, sputum,
or wound specimens
• If present, indicate likely contamination
with skin or mucosal flora
• A new specimen should be requested when
numerous squamous epithelial cells present
Poor Sputum Specimen
• Numerous squamous cells observed (oval,
rounded)
• Suspect mucosal cells from oral tract
Good Sputum (LRT)
Specimen
• Lung epithelial cells (elongated)
• None or few squamous cells
Circumventing NF:
Invasive Procedures
• Allow physician to avoid NF when
collecting specimen:
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Transtrachael aspirate
Suprapubic aspirate
Bronchoscopy (bronchial wash)
Needle biopsies
Transtrachael Aspirate
Suprapubic Aspirate
Specimen Identification
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Patient’s name and ID number
Patient’s location
Patient’s physician
Site/source of specimen
Type of exam requested (bacteria,
fungus, virus, parasite)
• Tentative clinical diagnosis
• Date and time of specimen collection
• If antibiotics administered - type,
dosage and time
Specimen: Swab
• Convenient and economical, but:
• Often inadequate amount
• Recovery of bacteria usually <10% of
original innoculum
• Often used for throat cultures and
for cervical, vaginal and urethral
secretions
• Newer “Flocked Swabs” – increase
surface area, collect more sample
(fluid, cells)
• Should not be used:
– Pus or exudate is available
– Surgical specimens
– Anaerobe or Mycobacterium
Specimen: Devices,
Transport, Media
• Syringes – good for aspirates; needles plugged
with sterile stopper
• Tubes, bottles, and jars – sterile, leak proof
• Fecal transport systems – polyvinyl alcohol
fixative for preservation of fecal parasites
• Sexually transmitted diseases - best to
inoculate media directly at bedside of patient
or use swab/transport media to retain
viability
• Specimens for virus or anaerobe culturing
need appropriate transport media to retain
viability
Specimen Transport
• Promptly transport to lab
– Preserve viability fastidious MOs
– Prevent overgrowth rapidly growing bacteria
which may not be pathogen
• Sometimes refrigeration warranted
i.e. urine specimen
• Refrigeration kill some fastidious MOs
– Streptococcus pneumoniae (sputum)
– Neisseria gonorrhoeae (genital tract)
Blood Specimen
• Septicemia – organisms or their toxins
present and growing in blood
• Bacteremia - presence of organisms in
blood without causing infection
• For septicemia, 2-3 cultures collected by
venipuncture in a 24 hour period:
– Collect 20-30 mls for each culture
– Inoculate into media directly at bedside of
patient
Wound Specimen
• Best specimen is aspirate of pus or
exudate
• A swab is usually not a good way to
collect specimens from wounds
Sterile Body Fluids
• Meningitis and
encephalitis –
collect CSF via a
lumbar puncture
• Pleural, pericardial
and synovial fluid –
aspirate and
collect sufficient
amount
Respiratory Specimen
• Upper respiratory tract infection – a swab
is sufficient
• Lower respiratory tract infection – collect
sputum. Alternatively, may use:
– Transtracheal aspiration
– Bronchial wash
– Lung aspirate
Urinary Tract Specimen
• Clean voided midstream specimen to
limit NF
• Catherization
• Suprapubic aspiration of bladder or
kidney
GI and Genital Specimen
• Gastroenteritis – collect stool sample in
sterile container
• Intestinal parasite - three separate stool
specimens collected as some present
intermittently
• Genital tract infection – swab or aspirate
of exudate plus direct inoculation onto
media
Other Specimens
• Ocular infection – a swab is
sufficient
• Tissue specimen – by biopsy or
autopsy
Direct Gross Exam of
Specimen
• CSF – if cloudy indicates infection
• Sputum – color, consistency and odor gives clues
as to causative agent
– Clear=virus
– Greenish=bacteria
• Stool - mucous and blood is typical of dysentery
• Anaerobe – often foul odor
• Actinomycete - visible granules (which are
bacteria aggregates)
Microscopy Exam: Differential
Stain – Gram Stain
• Gram(+) or Gram(-)
• Oil immersion – shape of bacteria
• Low power - fungi, some parasites, WBCs (hallmark
of acute bacterial infection is numerous PMNs)
• Positive and negative controls always done
• Gram stain of direct smear provide important
information for some specimens, but useless for
others (Give examples of each)
• Important not to over interpret Gram stain result
Gram Stain of Bacillus
species (B+)
Gram Stain of
Staphylococcus aureus (C+)
Gram Stain of Neisseria
species (C-)
Gram Stain of Haemophilus
species (B-)
Differential Stain:
Acid-fast Stain
• Acid-fast and non Acid-fast MOs
• Clinically important for diagnosing TB
• Mycobacterium tuberculosis grows slowly,
may be 6-8 weeks before culture report
• Important to physician - if seen in direct
smear, start TB antimicrobial therapy
Special Stain:Spore Stain
• Spore structure formed by vegetative cell
under adverse conditions, for survival
• Position of spore may be diagnostically
important
• Bacillus, Clostridium
Capsule Stain
• Outer structure, carbohydrate or
protein; protect against host
phagocytosis
• Background and MO stained, capsule
left unstained
• Klebsiella pneumoniae
Trichrome Stain
• For permanent stained smears of
intestinal parasites
• Giardia lamblia (trophozoite)
Iron-hematoxylin Stain
• Another way to make permanent
stained smears of intestinal parasites
• Entamoeba histolytica (cyst)
Wright Stain / Giemsa Stain
• Stain for blood cells
• Parasites and bacteria in the blood
are seen
• Trypanosoma in a blood smear
India Ink Wet Mount
• For encapsulated yeastlike fungi,
capsule remains unstained
• Cryptococcus neoformans
Lactophenol Cotton Blue Stain
• Observe fungi
• Hyphae, conidia
10% KOH Wet Mount
• Observe fungi from skin scrapings
• KOH destroys epithelial cells without
harming fungal elements
Iodine Stain
• Used for examination of parasitic
helminths in stool
• Stained egg in fecal specimen
Class Assignment
• Textbook Reading: Chapter 6
Specimen Collection and Processing
• Key Terms
• Learning Assessment Questions