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Streptococcus and Enterococcus
gram + coccus, form chains, no endospores, aerotolerant
Catalase for Strepto and Entero
Negative, typical for cells that don’t use O2, key in distinguishing the two from micro and staphylo
Alpha Hemolysis
partial breakdown of red blood cells (incomplete lysis), brownish/greenish color
Beta Hemolysis
destroy red blood cells completely (complete lysis), clear/slightly yellow tinted zone B=BAD!
Gamma Hemolysis
don’t lyse red blood cells (no lysis), medium is red and opaque
Alpha Hemolysis Species Example
Streptococcus pneumoniae and "Viridans” strep (pneumonia/meningitis)
Beta Hemolysis Species Example
Streptococcus pyogenes (Strep throat)
Gamma Hemolysis Species Example
Enterococcus faecalis (could cause UTI or endocarditis)
Selective Media
Select and favor growth of specific desired microbial group and prevent growth of undesired species (inhibits them)
Differential Media
Allow separation of different types of microbes based on their ability to perform particular biochemical reactions
Staphylococcus and Micrococcus
clusters/tetrads, no endospores, halotolerant(ability to grow in high salt concen.), aerobic respiration, gram +
Staphylococcus O2 Class
Facultative, use O2 or ferment
Micrococcus O2 Class
Strict aerobes, non-fermenters
Catalase for Staphylo/Micro
Positive
Staphylo Species
Staphylococcus aureus, epidermidis, and saprophyticus
Micro Species
Micrococcus luteus
Mannitol Salt Agar
Selective medium for Staphylo and Micro containing 7.5% NaCl and differential medium for Staphylo to see the ability to ferment the sugar mannitol Positive for Staphylo = yellow Negative = pink
Novobiocin Assay
Antibiotic effective against some Staphylococcus species, S. saprophyticus is resistant to novobiocin while epidermidis and aureus are sensitive.
Coagulase Assay
clumping factors (grainy), S. aureus only major pathogen of Staphylococcus and only species to carry coagulase
Pseudomonas
rods, no spores, motile, strict aerobes, non-fermenting, gram - (not enteric), use O2 to get energy from glucose & produce acids
Pseudomonas aeruginosa
only pathogen species, opportunistic infections
O/F assay for Pseudomonas
only when O2 is present oxidize glucose to acid, O/F assay used to separate from enteric species, yellow color near O2 and green at the bottom of tube
O/F Assays
indicator is bromothymol blue, green is alkaline, yellow is acidic, enterics are yellow, glucose is carbon source
Enterobacteriaceae Family
rods, no spores, facultative, respiration OR fermentation, gram -
Enterobacteriaceae Family Examples
E. coli, Enterobacter, Citrobacter, Salmonella, Proteus, Shigella
Glucose/Lactose Fermentation - Enterics
ALL ferment glucose, only some don’t ferment lactose (Salmonella, Shigella, Proteus)
Coliform vs Non-Coliform
Lactose fermenters vs. non-fermenters
MacConkey Agar Selective Traits
Contains crystal violet and bile salts(lots of acid will precipitate bile salts and make medium opaque): favors gram -, inhibits gram +
MacConkey Agar Differential Traits
Lactose fermenters > acid production > red colonies (pink agar); Non-fermenters > no acid > white colonies (yellow agar), neutral red pH indicator from red to yellow as pH increases (6.4 to 8.4)
MacConkey Agar
peptone and NaCl nutrition, Pink Positive and yellow negative for lactose fermentation
Eosin Methylene Blue (EMB) Agar Selective Traits
slightly more inhibitory than MacConkey, selects gram - enterics over other gram - and gram +
Eosin Methylene Blue (EMB) Agar Differential Traits
Lactose fermentation detected by pH dyes of Eosin and Methylene Blue,
EMB positive results
Dark purple/black - enterobacter, metallic green (lots of acid) - E. coli
EMB negative results
colonies remain uncolored/translucent maybe light yellow-purple color
EMB agar
some samples won’t grow on EMB so preference is MacConkey for initial isolation, nutrition is gelatin protein
E. coli vs Enterobacter aerogenes on EMB
E. coli will produce metallic green colonies while aerogenes will produce dark colored colonies (both are still positive for lactose fermentation)
Phenol Red Carbohydrate Assay
detect fermentation of sugars by detecting acid end products, positive: yellow broth negative: red broth (beef extract and peptones)
Lysine Decarboxylase Assay
Decarboxylases are enzymes that remove the carboxyl group from amino acids, positive: light/dark purple (cells produced LD & cadaverine) negative: yellow (did not produce LD or C)
Methyl Red Assay
Differentiated based on small amount of organic acid produced vs large amounts by glucose fermentation with methyl red pH indicator
Methyl Red Assay Positive Result
Lots of acid is produced and becomes red broth (E. coli)
Methyl Red Assay Negative Result
minimal acid is produced and becomes yellow broth (E. aerogenes)
Citrate Assay
to determine if a cell uses citrate as its sole carbon source
Citrate Positive Result
Produces ammonia (alkaline end product) and will turn deep blue color (E. aerogenes)
Citrate Negative Result
medium is green with no cell growth (E. coli) layer of slime can lead to false positive
Urease Assay
the ability of bacteria to produce urease enzyme that breaks down Urea into ammonia
Urease Positive Result
bright pink broth within 24 hours means produces large amounts of urease rapidly (Proteus)
Urease Negative Result
broth remains unchanged/orange within 24 hours either the cells don’t produce urease or do so slowly
SIM Assay (Sulfide Production)
detect hydrogen sulfide, enteric group does not produce H2S (E. coli, Enterobacter), Positive = dark/black agar by needle Negative = pale yellow color
SIM Assay (Indole Production)
detects breakdown of amino acid Tryptophan to Indole, add Kovac’s reagent indole is extracted from the medium by butanol, Positive = bright cherry red Negative = yellow/unchanged
SIM Assay (Motility)
To detect flagella and motility Positive = turbidity or bloom Negative = around stab remains transparent or tan/white colony growth (almost all sulfide positive means motility positive)
E. coli vs Enterobacter aerogenes Assay Test Difference
Methyl Red Assay (E. coli Positive), Citrate Assay (E. coli negative), Indole Assay (E. coli Positive)
Bacterial Strain VS Bacterial Species
Strain/Variant is population of cells derives from a single cell (all cells from given strain are genetically identical), Species is a collection of different strains and share genetically stable traits
Enteropluri System how it works
A tube with 12 chambers that can be inoculated by touching a needle to a single isolated colony and draw the needle through the chambers
Interpreting Results of the Enteropluri System
scored based on positive/negative based on the color of medium and circled numbers (pos results) are added together which produces a biocode of 5 #s
What family of microbes is Enteropluri System used for
Enterobacteriaceae (enterics) family
Why is the Enteropluri System useful
To identify species and strains of the Enteric family while also being timely efficient
Why don’t we directly test water for pathogens
pathogenic cells are present in low concentrations, won’t multiply in water, difficult to isolate, and too many types of waterborne pathogens (too expensive and time consuming)
Indicator Species
Enteric family, looking for coliforms and fermentation of lactose
Indicator Species traits
adapted to gut environment, easy to detect/quantify, uncommon in uncontaminated water, present where intestinal pathogens are, population density = degree of fecal contamination
Presumptive test (water analysis)
favor growth of coliforms to detect rapid production of gas as end product of lactose fermentation, scored based on how many positive tubes out of 3 (bubble in tube = positive) to get MPN
Confirmed test (water analysis)
confirm gas producers in lactose broth using EMB plate, positive = dark colored colonies negative = uncolored colonies
Completed test (water analysis)
use darkly pigmented colony from EMB plate to inoculate another lactose broth and if gas produced than almost certainly a coliform
Pathogens/Diseases associated with meat
E. coli (some strains) and Salmonella
Hektoen Enteric Agar
tests for lactose/sucrose and H2S production to isolate Salmonella and Shigella
Coliform species Positive result HE
HE(sugar): colonies are orange/salmon color HE(H2S): colonies with black centers
Coliform Species Negative Result HE
HE(sugar): blue/green colonies HE(H2S): no black centers on colonies
Fermenters vs non-fermenters Enteric & Salmonella
Enterics = fermenters of lactose & sucrose Salmonella/Shigella = non-fermenters
Salmonella vs Shigella
Salmonella positive for sulfide (black center) and Shigella negative for sulfide