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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