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MICR 301 – Fall, 2011• 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 General Medical MicrobiologyMicrobiology
General Medical General Medical MicrobiologyMicrobiology
Specimen Collection and Specimen Collection and ProcessingProcessing
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• 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:– Transtrachael aspirate– Suprapubic aspirate– Bronchoscopy (bronchial wash)– Needle biopsies
Transtrachael Aspirate
Suprapubic Aspirate
Specimen Identification• 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