Biology 216 - Lect 40 - Diagnostic Micro)
 
The following is a very brief outline of diagnostic strategies 
	for infectious diseases. Labs will vary, 
	but fundamentally adhere to the following.
A. Sequence of diagnostic steps:
	- Clinical examination
	- Specimen collection (based upon clinical exam)
	- Specimen transport
	- Specimen examined and processed
	- Inoculation of primary isolation media
	- Identification of etiology
	- Antibiotic sensitivity
	- Information used in treatment
B. Specimens and primary isolation media by site:
	1. Respiratory tract
	    Throat swab - R/O Group A Strept, Haemophilus
			BAP, Chocolate
			R/O Gonococci
			Choc/Thayer Martin
	     Nasopharynx - R/O Strept, Staph, Coryne
			BAP, Loeffler, Tellurite
			R/O Bordetella
			BAP, Choc, Bordet-Gengou (w/methicillin)
		Lung (sputa/transtracheal aspirate) - early AM
			< 10 epithelia, > 25 WBC per 100x field
			R/O Strept pneumoniae, enteric, Haemophilus
			BAP, Mac, Choc
			R/O Mycobacteria, Nocardia
			Liquify, decontaminate
			Acid fast stain
			Lowenstein Jensen (or equivalent)
			R/O Anaerobe (aspirate)
			BAP, Mac, BAP (anaerobe), Thioglycollate
	2. UTI - Infection of kidney, bladder, urethra
		Clean catch, catheter, process within 1 hour
		Urine specimen
		- Calibrated loop to BAP, Mac, Mannitol salts
			Quantify, note RBC ghosts or casts, 
			PMN, etiology are important
		- R/O Leptospira
			Fletcher's media, 30 C
	3. Genital - urethral or vaginal exudate or swab (not cotton)
		- Rely heavily on clinical presentation
		- R/O Gonorrhea
			Direct exam for Gram negative intracellular diplococci
			Plate BAP and Mac in case of UTI
			Thayer Martin (chocolate with antibiotics)
		- R/O Chlamydia
			Inclusions, ELISA, Fl-Ab
		- R/O Syphilis
			Serology (Non-treponemal, then treponemal)
	4. Stool - Feces, rectal swab
		- R/O Salmonella, Shigella, Campylobacter, .....
		Highly selective media (for Salmonella, Shigella) 
			- XLD, HE, SS
		Moderately selective media - MacConkey, EMB....
		Enrichment broth (for Salmonella) - 
			GN broth, Selenite - then subculture
		BAP w/ antibiotics at 42 C microaerophilic 
			(for Campylobacter)
	5. Wound - aspiration preferred over swab
		Open wound - expect normal flora
		Closed wound - all considered as pathogen
		BAP, Mac, BAP (anaerobic), Thioglycollate
	6. Blood - if suspect bacteremia 
		(fever, high pulse, murmur, high WBC)
		- 3 samples taken at least 1 hour apart
		preferably from different areas)
		- Specimen - thorough skin disinfection
			Adult - 10 - 20 ml blood
			Infant - 1- 5 ml blood
		- Inoculate blood into 9x volume of broth
			(ex. 10 ml blood into 90 ml TSB, BHI)
		- Incubate these blood bottles
			One vented (aerobic)
			One unvented (anaerobic)
		- Inspect for signs of growth (turbid, hemolysis, gas, etc...)
		- Blind subculture -> BAP, chocolate, same anaerobic
		- R/O Brucella - include Brucella blood media
		- R/O Leptospira - include Fletcher's media -> 30 C.
	7. CSF - Spinal tap (lumbar puncture) - 10 ml
		Immediate transport (not on ice)
		Centrifuge, stain and culture pellet.
		Consider age of patient for suspect etiology
		R/O Neisseria, Strept, Haemophilus, enteric, 
			BAP, chocolate, Thio
C. Secondary media - inoculate other media day 2 based upon: 
		Colony morphology, gram stain, 
		pigment, odor, hemolysis, change in 
		differential media, direct tests 
		(Fl-Ab, catalase, oxidase, etc.)
D. Antibiotic sensitivity - MIC or Kirby-Bauer