Lecture 1 - Identification Methods

gram positive

techoic acid + thick peptidoglycan layer
retains crystal violet
stains purple/blue

gram negative

thin peptidoglycan layer
stains pink/red from Safranin counterstain

gram stain procedure

fix to slide w/ methanol or heat
flood w/ crystal violet
rinse
flood w/ Gram's iodine
rinse
wash w/ decolorizer (acetone)
rinse
flood w/ Safranin counterstain
rinse & dry

what do you report in stool gram stain

only PMNs

what do you report in penis gram stain

G- diplococci are indicative of N. gonorrhea

what do you report in sputum gram stain

screen for contamination w/ saliva

what organisms should you report from most body sites

gram negative cocci

Q score

differentiates between saliva and sputum
count squamous epithelial cells & PMNs/low power field

Nugent scoring

for vaginal smears to detect bacterial vaginosis
measures lactobacillus (normal), Gardnerella, and Mobiluncus

alpha hemolysis

partial hemolysis
green around/under colony

beta hemolysis

complete hemolysis
clear/yellowish zone around/under colony

gamma hemolysis

no hemolysis

chromogenic media

colored colonies ID specific organisms

titer

reciprocal of highest dilution producing a positive result
e.g. 1/16 dilution is the last positive result: titer = 16

southern blot

DNA
microbes, mutations

northern blot

RNA
mRNA

real-time PCR

reporter probe hybridizes to DNA
as Taq passes, it dislodges and separates the reporter & quencher, creating a signal
get signal as rxn occurs, so you can see how much amplification

RT PCR

reverse transcriptase PCR
for RNA viruses

multiplex PCR

uses more than one primer set
detects multiple targets

nested PCR

phase 1: target DNA amplified
phase 2: PCR products used as target for 2nd round of PCR w/ different primers

branched PCR

signal amplified, not DNA duplication
pre-amplifier binds target DNA
bDNA assembles on pre-amplifier to produce stronger signal

Cq value

when RNA seq starts doubling
anything below is noise, nonspecific fluorescence

MALDI-TOF

ID's organisms growing on media using mass spectrometry
biomolecules separated by mass to charge ratio (gene sequencing)
compare w/ library to determine organism