MT MLO cheat sheet

Blood culture sets: collect 2-3 sets from separate venipuncture sites before antimicrobials; each set = one aerobic plus one anaerobic bottle; adults 20-30 mL total, 8-10 mL per bottle to maintain the ~1:10 blood-to-broth ratio.

Rejection criteria for sputum: Bartlett's criteria - >25 neutrophils and <10 squamous epithelial cells per low-power field indicates acceptable lower respiratory quality; saliva-like specimens are rejected.

Sterile sites (CSF, blood, joint fluid, tissue, abscess aspirate) are never rejected for quantity; process all volumes. CSF for culture should not be refrigerated if Neisseria meningitidis or fastidious organisms are suspected.

Transport times: CSF and body fluids within 1 hour at room temperature; stool for bacterial pathogens within 2 hours or placed in Cary-Blair; urine refrigerated at 2-8°C or preserved if not plated within 2 hours.

Anaerobic specimens: acceptable are aspirates, tissue, and blood; unacceptable are swabs (surface contamination), urine, and sputum. Transport in anaerobic transport systems; do not refrigerate.

Timing for viral and molecular testing: swabs for respiratory viruses in viral transport media, refrigerated at 2-8°C if processed within 48-72 hours, otherwise frozen at -70°C; avoid repeated freeze-thaw cycles.

Blood culture contamination: coagulase-negative staphylococci, diphtheroids, Cutibacterium acnes, and viridans streptococci in a single set suggest contamination; two or more sets positive with the same organism suggest true bacteremia.

Urine collection: clean-catch midstream; indwelling catheters are sampled from a newly placed catheter or the sampling port after disinfection, never from the drainage bag. Boric acid preservation or refrigeration prevents overgrowth.

Parasite preservation: fresh stool for O&P; formalin-ethyl acetate concentration plus permanent stain (trichrome or iron hematoxylin); SAF (sodium acetate-acetic acid-formalin) allows concentration and permanent smears from one specimen.

MacConkey agar selects gram-negatives; lactose fermenters (E. coli, Klebsiella, Enterobacter) form pink colonies, non-fermenters (Pseudomonas, Acinetobacter) are colorless. Blood agar and chocolate agar support fastidious organisms; chocolate agar supplies X and V factors.

Catalase-positive, coagulase-positive gram-positive cocci in clusters = Staphylococcus aureus. Coagulase-negative staphylococci are catalase-positive and coagulase-negative; novobiocin resistance identifies S. saprophyticus.

Streptococcal classification: bacitracin-susceptible, PYR-positive, bile esculin-positive = group A (S. pyogenes); hippurate-positive, CAMP-positive = group B (S. agalactiae); bile esculin-positive, 6.5% NaCl-positive = enterococcus.

Oxidase test: positive in Pseudomonas, Neisseria, Aeromonas, and Vibrio; negative in Enterobacterales and Acinetobacter. Indole-positive, citrate-positive, urease-positive, oxidase-negative = Klebsiella pneumoniae.

CLSI disk diffusion (Kirby-Bauer): use Mueller-Hinton agar, 0.5 McFarland inoculum, 150 mm plate with no more than 12 disks, incubate 16-18 hours at 35±2°C, measure zone diameter in mm and interpret with current CLSI breakpoints.

MIC methods: broth microdilution is the reference method; Etest produces a gradient MIC; automated systems require supplemental testing for inducible resistance and for organisms with known testing issues.

MRSA: cefoxitin 30 µg disk or oxacillin MIC; mecA-mediated resistance. Inducible clindamycin resistance (D-test) is reported when erythromycin-resistant, clindamycin-susceptible isolates show blunting of the clindamycin zone.

ESBL confirmation: ceftazidime and cefotaxime tested alone and with clavulanate; a ≥5 mm increase in zone or ≥3 two-fold dilution decrease in MIC confirms ESBL. Carbapenemase testing (mCIM/eCIM) is used for carbapenem-resistant Enterobacterales.

AmpC (SPACE organisms: Serratia, Pseudomonas, Acinetobacter, Citrobacter, Enterobacter): cefoxitin resistance plus third-generation cephalosporin resistance suggests inducible AmpC; cefepime and carbapenems are stable.

Beta-lactam susceptibility testing for Streptococcus pneumoniae uses oxacillin 1 µg disk as a screen; penicillin MIC by Etest or microdilution is required for confirmation and for meningitis breakpoints.

Specimen processing for mycobacteria: NALC-NaOH digestion-decontamination (2-3% NaOH final) followed by neutralization and concentration at 3000 × g for 15 minutes; inoculate solid (LJ, 7H10/7H11) and liquid (MGIT) media.

Acid-fast staining: Ziehl-Neelsen (hot carbolfuchsin) or Kinyoun (cold) stain; auramine-rhodamine fluorochrome stain is more sensitive for screening; mycobacteria appear red (ZN/Kinyoun) or yellow-green fluorescent.

M. tuberculosis complex identification: niacin positive, nitrate reduction positive, catalase negative at 68°C, slow grower (2-6 weeks on solid media); confirm with nucleic acid amplification (Hain line probe, GeneXpert MTB/RIF).

Nontuberculous mycobacteria: rapid growers (M. fortuitum, M. chelonae, M. abscessus) grow within 7 days; M. avium complex is slow-growing, nonpigmented, and often requires broth culture for isolation.

Nocardia: gram-positive, weakly acid-fast (modified Kinyoun), branching filamentous rods; aerobic, grows on blood agar and Sabouraud agar; colonies are chalky and may be orange; colonies are slow (3-7 days).

Nocardia identification: catalase-positive, urease-positive, nitrate reduction variable; susceptibility testing requires broth microdilution per CLSI with defined breakpoints; TMP-SMX is commonly tested.

Mycobacterial susceptibility: proportion method on 7H10 agar (1% proportion = resistant); liquid MGIT 960 SIRE for first-line drugs; molecular testing for rifampin resistance (rpoB) and isoniazid resistance (katG, inhA).

Safety: mycobacterial work is performed in a BSL-2 cabinet with BSL-3 practices for M. tuberculosis complex; Nocardia is handled under BSL-2 with aerosol precautions.

Sabouraud dextrose agar (SDA, pH 5.6, with chloramphenicol and gentamicin) is used for fungal isolation; incubate at 25-30°C for molds and 35-37°C for yeasts; hold mold cultures up to 4 weeks.

Yeast identification: germ tube test (Candida albicans and C. dubliniensis positive in 2-3 hours); cornmeal-Tween 80 agar for chlamydospores and pseudohyphae; chromogenic agar for presumptive species; MALDI-TOF and DNA sequencing for definitive ID.

Candida glabrata: germ tube negative, no pseudohyphae, small yeast cells; intrinsically resistant to fluconazole (high MIC) and often echinocandin-susceptible; C. krusei is intrinsically resistant to fluconazole.

Cryptococcus neoformans: encapsulated yeast, positive India ink, positive latex agglutination for capsular antigen, urease-positive, phenol oxidase-positive (bird seed agar browning); grows at 37°C.

Dimorphic fungi: Histoplasma capsulatum (tuberculate macroconidia at 25°C, yeast at 37°C), Blastomyces dermatitidis (broad-based budding yeast), Coccidioides immitis (arthroconidia), Sporothrix schenckii (cigar-shaped yeast).

Aspergillus: septate hyphae with acute-angle (45°) branching; Aspergillus fumigatus grows at 45°C; Aspergillus niger has dark conidia; galactomannan antigen is a serum marker.

Mucorales (Rhizopus, Mucor, Lichtheimia): broad, nonseptate (pauciseptate) hyphae with right-angle branching; culture rapidly produces woolly colonies; treatment is amphotericin B or posaconazole.

Dermatophytes: Trichophyton, Microsporum, Epidermophyton; identified by macroconidia morphology and biochemical tests (urease, hair perforation, trichophyton agar); KOH with calcofluor white for direct exam.

Antifungal susceptibility: CLSI M27 (yeasts) and M38 (molds) broth microdilution; echinocandins are not routinely tested for molds; Cryptococcus is not routinely tested because of poor correlation.

Stool O&P: direct wet mount in saline (motile trophozoites) and iodine (cysts); concentration by formalin-ethyl acetate; permanent trichrome stain for morphology; examine at least three specimens on alternate days.

Entamoeba histolytica vs. E. coli: E. histolytica trophozoites have ingested RBCs, a single nucleus with central karyosome; cysts have 1-4 nuclei and blunt chromatoid bars; E. coli cysts have 8 nuclei.

Giardia lamblia: pear-shaped trophozoite with two nuclei ("owl eyes"), ventral sucking disk, and falling-leaf motility; cyst is oval with 4 nuclei; antigen EIA and DFA are sensitive alternatives.

Cryptosporidium and Cyclospora: modified acid-fast stain (Kinyozi) or auramine; Cryptosporidium oocysts are 4-6 µm, Cyclospora 8-10 µm; Cystoisospora 20-30 µm; UV autofluorescence is used for Cyclospora and Cystoisospora.

Blood parasites: thick and thin Giemsa-stained films; thick film for detection, thin film for species identification; Plasmodium falciparum shows multiple rings, appliqué forms, and banana-shaped gametocytes; P. vivax shows Schüffner's dots and enlarged RBCs.

Babesia: intraerythrocytic rings and Maltese cross tetrads; no pigment; distinguish from Plasmodium by absence of hemozoin and lack of schizonts in peripheral blood.

Microfilariae: Knott's concentration or membrane filtration; stained blood films distinguish species by sheath presence and tail nuclei; Wuchereria bancrofti has a sheath and no tail nuclei.

Serology and molecular: ELISA for Strongyloides and Toxocara; O&P is insensitive for Strongyloides, so agar plate culture or Baermann concentration is recommended; PCR panels detect Giardia, Cryptosporidium, and Entamoeba.

Pinworm: cellulose tape (Scotch tape) preparation from perianal skin; eggs are oval and flattened on one side; stool examination is insensitive.

Cell culture: primary cells (monkey kidney), diploid fibroblasts (MRC-5, WI-38), and continuous lines (Hep-2, HeLa, Vero); observe for cytopathic effect; shell vial centrifugation-enhanced culture with monoclonal antibody staining detects viruses within 24-48 hours.

Respiratory virus testing: multiplex RT-PCR panels detect influenza A/B, RSV, SARS-CoV-2, adenovirus, and rhinovirus; nasopharyngeal swabs in viral transport medium are the preferred specimen.

HIV testing algorithm: fourth-generation antigen/antibody immunoassay; reactive results reflex to HIV-1/HIV-2 antibody differentiation; HIV-1 nucleic acid test resolves indeterminate results; confirm with Western blot only if NAT is unavailable.

Hepatitis serology: HBsAg indicates infection; anti-HBs indicates immunity; anti-HBc IgM indicates acute infection; HCV antibody is a screening test and HCV RNA confirms active infection.

Molecular methods: real-time PCR with Ct values; multiplex PCR panels; nucleic acid sequence-based amplification; sequencing for resistance (e.g., HIV pol gene, SARS-CoV-2 variants); line probe assays for HPV genotyping.

Quality controls for molecular assays: positive and negative controls each run; extraction control to monitor inhibition; contamination control with uracil-N-glycosylase and unidirectional workflow; separate pre- and post-amplification areas.

Viral load monitoring: HIV-1 RNA, HCV RNA, and CMV DNA by quantitative PCR; report in copies/mL or IU/mL; the WHO international standard allows comparison across assays.

Viral transport media: Hanks' balanced salt solution with bovine serum albumin, antibiotics, and antifungal agents; do not use calcium alginate swabs (inhibit PCR and culture); flocked swabs release more sample.

CLIA '88 QC: two levels of control (normal and abnormal) for each quantitative test each day of testing; for qualitative tests, a positive and negative control; for tests performed less than daily, controls are run on each day of testing.

Levey-Jennings chart rules (Westgard): 1-2s warning, 1-3s rejection, 2-2s rejection, R-4s rejection, 4-1s and 10x rejection rules; systematic errors show shifts or trends, random errors show scatter.

Calibration and verification: calibration is verified with at least two levels of control; method verification includes accuracy, precision, reportable range, and reference interval; new lots of reagents are verified against old lots.

Proficiency testing: required for CLIA-regulated analytes; results must be submitted to the PT provider by the deadline; failures require corrective action and may trigger a plan of correction.

Biosafety levels: BSL-1 standard precautions; BSL-2 for most clinical specimens, with biosafety cabinet for aerosol-generating procedures; BSL-3 for M. tuberculosis and Brucella; BSL-4 for Ebola and Marburg.

Chemical safety: SDS (Safety Data Sheet) on file for every hazardous chemical; eyewash and safety shower within 10 seconds' travel; formaldehyde, xylene, and glutaraldehyde are common hazards; monitor with exposure badges.

Fire safety: PASS (Pull, Aim, Squeeze, Sweep) for extinguishers; know the location of alarms, exits, and fire blankets; RACE (Rescue, Alarm, Contain, Extinguish/Evacuate) for response.

Sharps and spills: sharps in puncture-resistant containers; spill cleanup with absorbent material, disinfectant (10% bleach, 1:10 dilution of 5.25% sodium hypochlorite), and proper disposal; report exposures per OSHA bloodborne pathogens standard.

CLIA complexity categories: waived, moderate, and high complexity; laboratories must have a CLIA certificate, a laboratory director, and documented personnel qualifications for each testing category.

Personnel requirements: a laboratory director for high-complexity testing must hold an MD/DO with board certification or a PhD in a chemical, physical, biological, or clinical laboratory science with relevant experience; general supervisor and testing personnel qualifications are specified per category.

CAP and Joint Commission accreditation: CAP inspections every two years; CAP requires a quality management program, competency assessment of personnel at least semiannually for the first year and annually thereafter, and documented corrective action.

OSHA bloodborne pathogens standard: exposure control plan, hepatitis B vaccination offered, personal protective equipment, engineering controls, post-exposure evaluation, and annual training.

Quality indicators: turnaround time, specimen rejection rate, contamination rate for blood cultures, corrected reports, and proficiency testing performance; monitor and trend these metrics.

Budget and staffing: workload units (minutes per test) determine staffing; productivity = tests per FTE; cost per test includes reagents, labor, and overhead; evaluate send-out versus in-house testing.

Competency assessment elements: direct observation, review of test results and records, blind sample testing, and assessment of problem-solving skills; required for all testing personnel.

Education and training: documented training checklists, annual safety training, continuing education credits for certification maintenance, and a structured orientation for new employees.