unit 1 microbio

Empiric antimicrobial prescribing

before pathogen ID, broad spectrum, Under treatment (ineffective, cost, mortality, length of stay), Over-treatment (antimicrobial resistance, cost, organ toxicities, depletion of normal flora, C. difficile infections)

direct specimen vs microorgan culture testing types

d- Immunoassay Methods, Molecular Diagnostics, stains

c- Screening vs Comprehensive Cultures

rapid diagnostic testing RDT

Direct specimen testing, tests colony growth, detects antimicrobial resistance genes, Immunoassays, Molecular diagnostics, MALDI-TOF Mass Spect

direct specimen testing

STAT results, specimen came directly from patient not culture grown from specimen, determines specimen quality, cell types, and bacteria/yeast

gram stain

detects cells (high wbcs means infectious process) and bacteria/yeast (purple means gram pos)

direct testing antigen detection- immunoassay

known antibody in the test detects the unknown ag in patient, enzyme or substrate visualizes reaction with color, Streptococcus pneum ag test on urine, throat swab

direct testing serology (serum)

detects immune response to specific infectious disease agent, measures patient ab response to ag, positive weeks after primary infection, IgM = recent infection, IgG (4x increase from acute to recovery- convalescent titer

direct specimen testing- molecular detection

detects dna or rna unique to specific organism, mycobacterium tuberculosis, West nile virus WNV

types of culture methods

culture growth, phenotypic testing (physical characteristics to make ID not gen material), antimicrobial susceptibility testing AST

susceptibility testing

inoculate a specimen to various media, incubate 18-24 hours, 35-37 C, grow bacteria/yeast in specimen

screening culture

report only specific path from specific source, throat (group A step coc), gonorrhea (Neisseria gonorrhoeae)

comprehensive culture

report all that is growing (path and normal flora), sputum, urine, wound, csf, blood

Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM)

appropriately and accurately use of laboratory tests for the diagnosis of infectious diseases for all healthcare workers to follow

disinfection

destroys path organisms not spores, physical (boiling, pasteurize, UV light), chemical (phenol, alc, detergents, bleach), diff work areas (cabinet and hood)

sterilization

all forms of microb life destroyed

sterialization- incineration

dispose of infectious waste

sterilization- dry heat

sterilize wire loop with bacti-cinerator, glassware, oil, petroleum, powders

sterilization- moist heat (autoclave)

15lbs/in 15 min, 121 C, surgical bandages, instruments, water, contaminated materials and media

sterilization- filtration liquids

thin membrane filters retain microorganisms, serum, antibiotic solutions, toxic chemicals radioisotopes, vaccines, carbs

sterilization- filtration air

HEPA filter, isolation room, operating room, biological safety hood, respirator

sterilization QC procedure

varies on temp, time, indicator strip per load, check bacterial spores weekly, check gauges and recalibrate semiannually

when bleach

workstation after each use

handling patient specimen

process under bio safety hood

slide 2

specimen process

collect quality specimen for quality data, transport specific to each specimen (protects wbcs, bacteria/yeast with no growth)

specimen collection timing and container

collected during acute phase BEFORE administration of antibiotics, sterile (except for stool- clean and leak proof

specimen collection test requisition

Name, age, sex, location, physician’s name, specific anatomic site, date and time of collection, clinical diagnosis or relevant history, antimicrobial agents patient is on (if applicable)

specimen collection- Site of infection

speciemn type (blood, body fluid, tissue), sterile vs non sterile

specimen collection- labeling

Patient name, ID number, room number, culture site/source, date and time of collection

preservatives/transport media

urine- boric acid

stool- cary-blair (formalin/polyvinyl alc PVA)

blood- sodium polyanethol sulfonate SPS

swabs- stuarts or amies transport medium, JEMBEC system

specimen transportation

asap, is delayed (fidge, freeze if more than 4 days, transport media, via mail regulations)

specimen processing workload prioritization

1st- critical and invasive specimen (fluids, brain, heart valves)

2nd- unpreserved specimen (tissue, sputum, feces)

3rd- Quantification specimen (catheter tip, urine, tissue)

4th- preverved specimen

specimen processing- criteria for rejection

obtain new before discarding old, note report condition of specimen, transport time over 2 hours, expectorated sputum submitted with more than 25 epithelial cells / LPF, QNS (insufficient quantity)

specimen processing- macroscopic examination of specimen

document physical characteristics, determine if special setup requirements are met

examples of notes on specimen processing reports

swab or aspirate, stool consistancy, blood or mucous present, volume of specimen present, fluid clarity (clear cloudy)

culture set up

some require special prep before culturing, concentratuin (centrifuge prior to plating to increase recovery bacteria), filtration (water analysis), homogenization (tissue grinding), decontaimination (kills normal flora in a specimen to help locate specific organism, mycobacteria)

capillary, PICC or central line blood collection

c- by pricking the skin

picc- drawn from a peripherally inserted central catheter

catheter vs supranat collection of urine

tube from bladder

aspiration from bladder via needle

selection of culture media

promote growth of all possible pathogens in specimen, proper pH, nutrients, moisture, standardized, in procedure, one or more plates

media types- nutrient

contains generic nutrients to enable growth of most nonfastidious (doesn’t need extra help) organisms, sheep blood agar SBA, blood agar plate BAP

media type- enriched

Contains complex substances (blood, serum, hemoglobin) or growth factors for the needs of specific species (fastidious), BAP, Chocolate agar CHOC (heated lysed blood and other nutrients)

selective media

Inhibits the growth of some organisms while allowing the growth of others, MacConkey MAC (inhib Gram-pos, pro Gram-neg), Phenylethyl alc agar PEA and columbia colistin nalidixic acid agar CNA (inhib neg, pro pos), Mannitol salt agar MSA (pro staphylococcus)

differential media

demonstrate visual characteristics of organisms (colony size, change in color, formation of gas or precipitates), BAP- hemolysis, MAC- lactose fermentation pink GNR colony, clear if not

selective and differential

MAC

enrichment broth media LIM

encourages growth of small numbers of a particular organism suppressing others

Thioglycolate broth THIO

Used in combination with growth on agar plates to detect small numbers of most aerobes, anaerobes, and microaerophiles

BAP purpose

enriched and differential

CHOC purpose

enriched

MAC purpose

selective and differential

CNA and PEA purpose

selective

streaking tech

isolation- 1st inoculate media with sterile loop, swab, pipette, non selective first, 2nd streak plate for isolated colonies in gram stains, biochem testing, and antimicrobial susceptibility testing

quantitation/colony count

lawn growth (criss cross)- Kirby-Bauer

inoculation step 1 and 2

1- label and date

2- inoculate plates with sterile loop/swab/pipette (first streak), choose thickest mucouses or bloody part, roll swab, 1st quadrand, close overlapping streaks, no need to redip between plates, non selective than selective

inoculation step 3

flame loop and make second streak, allow to cool first, 2nd quadrant by passing loop through edge of 1st quadrant 2-3 times

inoculation step 4

repeat step 3 for quadrant 3 and four

inoculation step 5 and 6

5- flame loop

6- repeat steps 3-5 for all plates

incubation of media- atmosphere

obligate aerobe (requires oxygen), microaerophile (requires reduced oxygen and increased CO2), facultative anaerobe (not deependent on oxygen but grows better with, bacterial path), capnophilic (requires increased CO2)

incubation of media- temp

psychrophiles (10-20 C, few pathogens), mesophiles (20-45 C, human pathogens and most bacteria), thermophiles (50-200 C, few pathogens), most are 35 C

incubation of media- pH

6.5-7.5, buffered media

incubation of media- moisture

evaporation concerns, 70-80% humidity

incubatior types

ambient air- room air, 35 C, high humidity

CO2- 5-10% CO2 mized with room air, 35C, high humidity

anaerobic- 5% hydrogen, 5-10% CO2, 85%-90% N2, 35C, high humidity

incubation length

moist- 48-72 hours

anaerobic cultures- 5-7 days

other- days-weeks

culture workflow first steps

compare specimen type with orders for specimen, find procedure, evaluate specimen acceptability, set up and process specimen, interpret and correlate results from all phases of testing

culture workup day 1

processing, setup and incubate plates/broth, perform direct specimen gram stain, preliminary report (direct specimen gram stain morphology)

culture workup day 2

colony morphology, perform colony Gram stain, perform biochemical testing, subbing of plates if needed, setup ID and susceptibility panel, preliminary report (org ID, quantitation if needed, next steps)

culture workup day 3

final report (org ID, quantitation, susceptibility results)

temp and equipment checks

daily for incubators, fridge, freezer, heating blocks, water baths

equipment- performance checks, maintenance (oiling, cleaning, replacing filters, recalibrating instruments)

CLSI clinical and lab standards institute

must QC all media, document performance and sterility, records maintained for 2 years

commercially prepped media : exempt

certificate of QC from manufacturer retained as long as it is in use, certain types of media still QC due to complexity/history of failing (CHOC selective for Neisseria, Campylobacter), inspect moisture sterility breakage appearance on lot shipments, user prepared documentation and sterility check

stains and reagents

QC based on procedure, pos and neg, typically tested day of use, documentation kept at least 2 years

antibiotics CLSI

controls of susceptibility testing, perform against specific strains of control organisms from the ATCC #, Variables can affect accuracy of susceptibility results (Antibiotic potency, agar depth (Kirby-Bauer test), evaporation (microtiter dilution), cation content, pH, thymidine content, instrument failure, inoculum concentration, temp, moisture (KB test), difficulty determining endpoints)

records of

tolerance limits, corrective action, procedures (temp, equipment, media, reagents, susceptibility testing, personnel)

gram stain

microscopic staining characteristics are used in the classification of microorganisms, color indicates reaction pos or neg, morphology, arrangment

stains used to visualize

bacteria, rbc, wbc, and epithelial cells

gram pos cell walls

purple/blue, bacilli or rod, cocci, lancet (elongated spherical), Low lipid content (thick cell wall of peptidoglycogen and techoic acid)

gram pos cocci GPC

single, pairs (lancet, elongated spherical shape), chains, clusters, tetrads (group of 4)

GPC staphylococcus

Divide in three alternating perpendicular planes, sister cells remain attached, grape like clustering

GPC streptococcus

divide in a single plane and sister cells remain attached, chains

GPC arrangement reporting

note singles, pairs, tetrads, clusters, or chains, capsules, predominant morphology (chains aka strep or clusters aka staph)

Gram pos bac or rod GPR GPBs

variable with spores, palisading (aligned side by side, Chinese letter pattern, club shaped, pleomorphic)

gram neg cell walls

re/pink, bac / rod, diplococci, coccobac, pleomorphic, high lipid (thin cell walls), Decolorizer effect: able to penetrate high lipid content cells, no arrangements

GNDC gram neg diplococci

Spherical bacteria with a slight indent (dip) into the cocci shape, bean shaped, short and fat, usually pairs, Neisseria or Moraxella, intracellular, capsules

GNCB gram neg coccobacilli

thin short rods almost spherical, some elongates pleomorphic, TINY, capsules, intracellular

gram neg rod bac GNR/B

spores only in pos, intracellular

pleomorphic

varies in size and shape

gram variable

pink and purple

gram stain steps

1. Primary stain: Crystal violet (purple) ~1 min

2. Rinse with water

3. Mordant: Gram’s Iodine (complexes with CV) ~ 1 min

4. Rinse with water

5. Decolorizer: acetone/alcohol (dehydrates lipids in cell wall – creates holes for CV-iodine to escape) ~2-3 seconds

6. Rinse with water

7. Counterstain: Safranin (pink) ~1 min

8. Blot (do not wipe) with paper towel

gram stain of colonial growth- culture plate

colonies are solid, label slide, place small drop of sterile saline to a slide, transfer a small portion of the colony to the slide using a sterile applicator stick and mix to emulsify, spread out to make a thin smear, air dry slides and heat fix for 1 min

gram stain of colonial growth- broth

label slide, mix sample, use sterile pipette to transfer 1-2 drops to the slide, spread out to make a thin smear using a sterile applicator stick, air dry and heat fix

yeast

gram pos, variable if over decolorized, larger than pos cocci

specimen gram stain cells

squamous epithelial cells, wbcs or polymorphonuclear PMNs (multiple lobed nucleus), rbcs, quantitation on LPF 10x

direct specimen gram stain process

quantify specimen, scan 10-30 feilds, move to concentrated area, interpretate the number and types of bacteria/organisms/cells (gram, shape, arrangement, intracellular?)

troubleshooting

over-decolorization, under decolorization, smear too thick or thin, organism too old, patient on antibiotics