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 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 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 -3 ml) If colony count >100 colonies (#MOs >10 5 /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 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 C atherization 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