Lab practical written section

when writing the name of an organism ALWAYS make sure it is

in italics

how to shorten name of organism

first letter of first word, period, full second word. (all in italics)

ex E. coli

total magnification equation

(Objective magnification) X (Ocular magnification) = Total magnification

ocular magnification is usally 10x

objective magnification can be 4x, 10x, 100x, etc

gram stain for a gram-negative species will appear

pink

gram stain for a gram-positve species will appear

purple

what are the 7 gram-negative tests we used

• Indole

• Methyl Red

• Voges Proskauer

• Simmons Citrate

• Oxidase

• EMB (Eosin methylene blue fermentation patterns)

• TSI

what medium is used for indole tests

tripticase soy broth

indole tests test for

Tests for presence of tryptophanase enzyme (breaks

down tryptophan into indole)

positive indole test

has pink/red layer on top

negative indole test

no color change (appears just yellow)

methyl red tests for

Tests for “mixed acid” vs “butanediol” fermentation

pathway usage (break down glucose)

Mixed acid pathway ends in

acidic end-products

Butanediol pathway ends in

neutral end-products

positive meythl red result (and what it means)

red color stays, meaning mixed acid pathway was used

negative meythl red result

copper color forms, meaning mixed acid pathway was NOT used. yellowish

Voges-Proskauer tests for

tests for positive presence of butanediol pathway (used to ferment glucose)

general rule for MR-VP testing

for a given organism, only one test will be positive!

VP positve results

color change to red

VP negative results

no color change (will stay pale yellow color)

Simmon’s Citrate agar contains

citrate as sole sugar source and bromothymol blue pH indicator

simmons citrate tests for

Testing for whether the organism can utilize citrate ALONE as a sugar source

positve citrate result

color change to blue

negative citrate result

no color change. stays green.

oxidase tests for

Testing for presence of cytochrome c oxidase enzyme

how long to wait for oxidase results

30 seconds (dont walk away! they will all change to purple if left long enough. waiting too long could lead to false positves)

positve oxidase result

color change to purple

negative oxidase result

no color change (stays white)

is EMB selective or differential?

both!

how to EMB plates work

dyes inhibit G(+)s, act as pH indicators for sucrose and lactose fermentation

(wealky selective. sometime G+ can grow)

if an organism is a Vigorous lactose/sucrose fermenters it will appear ______ on an EMB plate

black/purple (green if its E.coli)

if an organism is a slow lactose/sucrose fermenters it will appear ______ on an EMB plate

pink

if an organism does not ferment lactose/sucrose (not coliform) it will appear ______ on an EMB plate

clear

EMB tests for

lactose/sucrose fermentation

4 tests for gram-postive bacteria we used

• Bile Esculin

• Catalase

• Blood agar hemolysis patterns

• Mannitol salt agar fermentation patterns

Selective means

selects for certain bacteria, kills others

ex only G+ can grow on MSA

Differential means

differentiates between similar species

ex S. aureus turns yellow on MSA; S. epidermidis does not

is nutrient agar selective or differential?

neither!

is Mannitol Salt Agar (MSA) selective or differential

both

How does MSA work

High salt content SELECTS for G(+) species

Phenol red (pink color) turns yellow when mannitol sugar is digested, DIFFERENTIATING species

positve MSA result

yellow! indicates mannitol sugar digestion

negative MSA result

no color change. stays pink. no mannitol sugar digested

are blood agar plates (BAP) selective or differential

differential. DIFFERENTIATES species based on how the blood is/isn’t broken down

hard to grow organims can usually be grown on

BAP because bacteria love blood

BAP alpha hemolysis

partial breakdown. looks brown.

BAP beta hemolysis

total breakdown. looks yellow and has a clearish halo around it

BAP gamma hemolysis

no breakdown. just growth on top of plate; appears white/regualr color.

are Bile Esculin Slants (BES) selective or differential

both

how do BES work

SELECTS for G(+) species with bile salts

DIFFERENTIATES species by +/- esculin hydrolysis esculin by-products react with ferric citrate, turn media black

black BES means

esculin hydrolysis occured

Catalase Test determines

Determines if a species produces the enzyme catalase

positve catalse test

bubbles when hydrogen peroxide is added

negative catalse test

no bubbles

cell

A single, independent living unit

colony

A visible grouping of cells on a solid medium, all descended from a single original cell, creating a clone population.

culture

The practice of growing cells in a controlled environment, often referring to a population of cells derived from a single colony

aseptic technique encompasses

Methods of working free of microbial contamination

purpose of aseptic technique

Prevent culture/specimen contamination

Prevent contamination of yourself/others/environmen

examples of aseptic tenchique

Safely dispensing culture media

Flame-sterilizing instruments

Proper labeling, handling, and storing

Practicing correct hand hygiene and workspace disinfection

simple stains

coloring cells to see shape

gram stains

differentially coloring cells to see shape and determine Gram class

endospore stains

differentially coloring to see if the species makes spores

kirby bauer test is used to idenfity

an organisms antibiotic resistence proflie

how to interpret kirby bauer results

measure diamter and compare it to a suscetpibility table

• Susceptible—zone of inhibition falls in the susceptible range

• ▪ Resistant—zone of inhibition falls in the resistant range

• ▪ Intermediate—borderline response

Collected data and present in an antibiogram

Obligate aerobe:

needs O2 to live, breaks down sugars using it

Obligate anaerobe:

dies in presence of O2 (doesn't use it, can't detoxify it!)

Facultative anaerobe:

prefers O2 (more energy!), but can do anaerobic respiration and detoxify O2 when needed

Microaerophile:

can only stand O2 in low concentrations (uses it a little in metabolism, can only detoxify a little)

Aerotolerant:

doesn’t use O2 , but doesn’t die when exposed to it (can detoxify!)