Micro 202 Lab Exam 3

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