Micro- unit 4 GI SLG

Fresh Stool (Unpreserved)

Preferred for direct microscopic examination (fecal leukocytes, motile trophozoites) and rapid viral antigen/toxin detection

-must reach the lab within 1-2 hours of collection to prevent bacterial pH changes and parasite degeneration

Cary-Blair Transport Medium

essential preservation medium for routine bacterial stool cultures when transport delays exceed 2 hours

-Maintains viability of fastidious pathogens including Campylobacter jejuni, Shigella spp., Salmonella spp., Vibrio cholerae, and Yersinia enterocolitica. 

Parasitology Preservatives (Two-Vial / Single-Tube Systems)

10% Neutral Buffered Formalin preserves protozoan cysts, helminth eggs, and larvae for concentration techniques.

-Polyvinyl Alcohol (PVA) or mercury-free zinc/copper substitutes fix protozoan trophozoites and cysts for permanent trichrome staining.

-Sodium Acetate-Acetic Acid-Formalin (SAF) and single-tube commercial systems accommodate both concentration and permanent staining.

Rectal Swabs

Reserved primarily for pediatric patients or outbreak screening (e.g., Shigella or VRE surveillance).

-Swabs must show visible fecal staining to ensure adequate sampling.

Duodenal Aspirates / String Test (Entero-Test)

Specialized collection methods for diagnosing upper small intestinal parasites such as Giardia lamblia and Strongyloides stercoralis larvae when stool examinations remain negative. 

Unpreserved stool > 2 hours old; un-refrigerated viral/toxin samples. 

Bacterial overgrowth of normal commensals alters stool pH and kills fastidious pathogens (Shigella, Campylobacter).

-Reject unpreserved stool > 2h old.

Presence of barium, bismuth, mineral oil, urine, or toilet water contamination

Barium and mineral oil mask microscopic parasite morphology. Urine destroys motile protozoan trophozoites.

-Reject and request specimen collected 7-10 days post-barium

Routine stool culture submitted on patients hospitalized

Low yield for conventional bacterial enteric pathogens (Salmonella/Shigella).

-Reject routine culture; recommend Clostridium difficile toxin testing instead.

macroscopic examination of stool consistency watery, loose, formed

dictates direct wet prep procedures

macroscopic evaluation of Liquid/watery stool consistency

prioritized for motile trophozoites (Giardia, E. histolytica

macroscopic evaluation of formed stools

protozoan cysts and helminth ova

Methylene Blue & Gram Stain

Staining mucosal mucus with Loeffler's methylene blue or Gram stain allows quantitation of polymorphonuclear neutrophils (PMNs).

Invasive Pathogen Correlation

Presence of abundant fecal PMNs correlates with invasive bacterial colitis caused by Shigella spp., Salmonella enterica, Campylobacter jejuni, Invasive E. coli (EIEC), or Clostridium difficile. 

Non-Invasive / Viral Etiology

Absence of fecal PMNs (mononuclear cells or red cells only) is characteristic of toxigenic watery diarrhea (Vibrio cholerae, ETEC), viral gastroenteritis (Norovirus, Rotavirus), or non-invasive parasitic infections (Giardia lamblia). 

saline wet mount target organisms and structures

Motile protozoan trophozoites (Giardia, E. histolytica), helminth eggs & larvae. 

saline wet mount diagnostic utility and key characteristics

Unpreserved fresh liquid stool. Demonstrates characteristic progressive motility (E. histolytica) or falling-leaf motility (Giardia). 

iodine wet mount target organisms and structures

Protozoan cysts, nuclear details, glycogen vacuoles, helminth ova. 

iodine wet mount diagnostic utility and key characteristics

Lugol's or D'Antoni iodine stains nuclei dark brown and highlights chromatoid bodies and glycogen stores for cyst differentiation. 

trichrome stain target organisms and structures

Intestinal protozoan trophozoites and cysts (E. histolytica, E. coli, Giardia, Dientamoeba). 

trichrome stain diagnostic and key characteristics

Permanent stain. Background stains green/blue;

-protozoan cytoplasm stains blue-green; nuclear chromatin and ingested RBCs stain dark red/purple. 

Modified Acid-Fast (Kinyoun) target organisms and structures

Intestinal Coccidia: Cryptosporidium parvum, Cystoisospora belli, Cyclospora cayetanensis. 

modified acid fast (kinyoun) diagnostic utility and key characteristics

Oocysts resist decolorization and stain bright pink-red against a blue-green background.

-Cryptosporidium oocysts measure 4-6 µm. 

MacConkey (MAC) Agar selective and differential agents

Bile salts, crystal violet (inhibits Gram-positives); Lactose, Neutral Red indicator. 

MAC agar target pathogens and colony morph

Enterobacteriaceae. Lactose fermenters (pink/red); Non-lactose fermenters (NLF - clear/colorless: Salmonella, Shigella, Yersinia). 

Hektoen Enteric (HE) agar target pathogens and colony morphology

Salmonella (blue-green colonies with black H2S centers); Shigella (green colonies without H2S); Coliforms (salmon/orange). 

HE agar selective and differential

Deoxycholate (inhibits Gram-positives); Xylose, Lysine, Lactose, Sucrose; Phenol Red; Thiosulfate. 

SMAC selective and differential agents

Standard MAC formulation replacing lactose with D-sorbitol. 

SMAC target pathogens and colony morphology

E. coli O157:H7 (STEC). Sorbitol-negative (colorless/clear colonies at 18-24h);

-normal E. coli ferments sorbitol (pink). 

Campy-Blood Agar (Campy-BA) / CVA selective and differential agents

Brucella agar base, 5-10% sheep blood;

-Vancomycin, Trimethoprim, Polymyxin B, Amphotericin B, Cefoperazone. 

Campy-Blood Agar (Campy-BA) / CVA target pathogens and colony morphology

Campylobacter jejuni / C. coli. Runny, spreading, non-hemolytic grey colonies.

-Requires 42°C microaerophilic incubation. 

TCBS agar selective and differential agents

Thiosulfate, Citrate, High Bile Salts (pH 8.6); Sucrose; Bromothymol blue indicator.

TCBS agar target pathogen and colony morphology

Vibrio spp. Vibrio cholerae ferments sucrose (yellow colonies);

-Vibrio parahaemolyticus & V. vulnificus are sucrose-negative (green colonies). 

CIN agar selective and differential agents

Cefsulodin, Irgasan, Novobiocin; Bile salts, Crystal violet; Mannitol, Neutral Red. 

CIN agar target pathogens and colony morphology

Yersinia enterocolitica. Typical 'bull's-eye' colonies (dark pink/red center with translucent outer border) after 24-48h at 25°C-30°C.

Selective Enrichment Broths

Selenite F Broth and Gram-Negative (GN) Broth suppress normal coliform overgrowth during the first 6-12 hours of incubation, enhancing recovery of low numbers of Salmonella and Shigella from stool.

Microaerophilic Atmosphere (Campylobacter)

Requires 5% O2, 10% CO2, and 85% N2 at 42°C. Incubation at 42°C inhibits normal enteric flora and non-pathogenic Campylobacter species. 

Ambient / Capnophilic Atmosphere (Routine Enterics & Yersinia):

Routine enteric plates (MAC, HE, XLD, SMAC) incubate in ambient air at 35°C-37°C.

-CIN agar for Yersinia incubates optimally at room temperature (25°C-30°C) for 48 hours. 

Escherichia coli (STEC/EHEC) key biochemical and diagnostic profile

NLF on SMAC (sorbitol -)

MUG negative

Shiga toxin 1 & 2 positive by EIA/PCR

Indole +. 

Escherichia coli (STEC/EHEC) clinical significance and disease profile

Hemorrhagic colitis, bloody diarrhea, Hemolytic Uremic Syndrome (HUS). O157:H7 serotype.

Salmonella enterica key biochemical and diagnostic profile

TSI K/A,

Gas +,

H2S +;

LIA K/K (lysine decarboxylase +);

Urea -;

Citrate +;

Indole -;

Serotyping +.

salmonella enterica clinical significance and disease profile

Gastroenteritis, enteric/typhoid fever (S. Typhi: minimal H2S ring, no gas), bacteremia. 

Shigella spp. key biochemical and diagnostic profile

TSI K/A,

Gas -,

H2S -;

LIA K/A (no decarboxylation);

Urea -;

Non-motile;

Serogroups A, B, C, D. 

shigella sp. clinical significance and disease profile

Bacillary dysentery (bloody, mucoid stool with abundant PMNs). Very low infectious dose (<100 organisms).

Yersinia enterocolitica key biochemical and diagnostic profile

Bull's-eye on CIN; TSI A/A (sucrose fermentation); Urea +; Motile at 25°C, Non-motile at 35°C. 

Yersinia enterocolitica clinical significance and disease

Enterocolitis, mesenteric adenitis (pseudoappendicitis), transfusion-associated bacteremia. 

campylobacter jejuni key biochemical and diagnostic profile

Curved/gull-wing Gram-neg rod;

Darting motility;

Oxidase +;

Catalase +;

Hippurate hydrolysis +.

campylobacter jejuni clinical significance and disease

leading cause of bacterial gastroenteritis; bloody diarrhea; associated with Guillain-Barré syndrome. 

vibrio cholerae key biochemical and diagnostic profile

Comma-shaped Gram-neg rod;

Oxidase +;

TCBS yellow (sucrose +);

String test +;

Halotolerant (0% NaCl). 

vibrio cholerae clinical significance and disease profile

Cholera (profuse 'rice-water' watery diarrhea, severe dehydration). Cholera toxin mediated. 

Vibrio parahaemolyticus / vulnificus key biochemical and diagnostic profile

Oxidase +;

TCBS green (sucrose -);

Requires NaCl for growth (halophilic).

V. vulnificus lactose +.

Vibrio parahaemolyticus / vulnificus clinical significance and disease profile

Seafood-associated gastroenteritis, wound infections, fulminant septicemia in liver disease (V. vulnificus). 

Aeromonas hydrophila key biochemical and diagnostic profile

Gram-neg rod;

Oxidase + (diff from Enterobacteriaceae);

Beta-hemolytic on SBA;

DNase +. 

Aeromonas hydrophila clinical significance and disease profile

Waterborne gastroenteritis, wound infections following freshwater exposure. 

Clostridium difficile key biochemical and diagnostic profile

Spore-forming Gram-pos rod;

Toxin A (enterotoxin) & Toxin B (cytotoxin) positive via EIA/PCR. 

Clostridium difficile clinical significance and disease profile

Pseudomembranous colitis, antibiotic-associated diarrhea. Spore control required. 

Entamoeba histolytica key biochemical and diagnostic profile

Cyst: 10-20 µm, 1-4 nuclei, central karyosome, chromatoid bars.

Troph: ingested RBCs. 

entamoeba histolytica clinical significance and disease profile

Amebic dysentery, liver abscesses. Differentiated from non-pathogenic E. dispar.

Selective Testing & Reporting (Salmonella & Shigella)

Primary agents include Ampicillin, a Fluoroquinolone (Ciprofloxacin), Azithromycin, and a 3rd generation Cephalosporin (Ceftriaxone)

-For extraintestinal Salmonella isolates, fluoroquinolones and cephalosporins must be tested and reported.

Contraindication / Withholding AST in STEC / EHEC

for Shiga toxin-producing E. coli (STEC / E. coli O157:H7).

-Antibiotic treatment induces bacterial lysis, triggering massive Shiga toxin release and dramatically increasing the risk of Hemolytic Uremic Syndrome (HUS).

Campylobacter Susceptibility Testing

AST is not performed routinely for uncomplicated C. jejuni enteritis.

-When clinically indicated (severe or immunocompromised cases), Kirby-Bauer disk diffusion or E-test using Mueller-Hinton agar with 5% sheep blood is performed for Macrolides (Erythromycin/Azithromycin - drug of choice) and Fluoroquinolones (Ciprofloxacin).

Extended-Spectrum Beta-Lactamases (ESBL)

plasmid-mediated enzymes (CTX-M, TEM, SHV) in E. coli, Salmonella, and Klebsiella that hydrolyze penicillins, cephalosporins (ceftriaxone, cefotaxime, ceftazidime), and aztreonam. Confirmed by clavulanic acid inhibition testing.

Carbapenem-Resistant Enterobacteriaceae (CRE)

production of carbapenemases (KPC, NDM, VIM, OXA-48) conferring resistance to all beta-lactams including carbapenems (ertapenem, meropenem, imipenem).

-Requires immediate infection control isolation. 

Fluoroquinolone Resistance (gyrA / parC Mutations)

Increasing chromosomal point mutations in DNA gyrase and topoisomerase IV confer high-level resistance to ciprofloxacin in Salmonella, Shigella, and Campylobacter isolates worldwide. 

Microscopic vs. Culture Alignment diagnostic synthesis and result correlation

Abundant fecal PMNs on direct smear must align with recovery of invasive bacterial pathogens (Shigella, Salmonella, Campylobacter, EIEC) or C. difficile toxin positivity.

-Absence of PMNs with watery stool points toward STEC, Vibrio, or viral gastroenteritis. 

Molecular Syndromic GI Panels diagnostic synthesis and result correlation

Multiplex PCR panels detect bacterial, viral, and parasitic targets directly from stool within hours.

-Positive molecular results for STEC, Salmonella, Shigella, or Campylobacter must be correlated with reflex culture for isolate recovery, serotyping, and public health submission.

Immediate Reportable Pathogens

isolation or molecular detection of Shiga toxin-producing E. coli (STEC), Salmonella enterica (especially S. Typhi), Shigella spp., Vibrio cholerae, Yersinia enterocolitica, and Listeria monocytogenes

Public Health Reference Isolate Submission

Isolates of STEC, Salmonella, Shigella, and Vibrio must be forwarded to state public health laboratories for confirmatory serotyping, pulsed-field gel electrophoresis (PFGE), or whole-genome sequencing (WGS) via PulseNet for outbreak tracking. 

Antibiogram & Infection Control

Cumulative enteric antibiogram data must be updated annually to track emerging resistance trends (e.g., fluoroquinolone-resistant Salmonella/Campylobacter) and assist antibiotic stewardship teams.