BIO 379 exam 1

Why do we incubate petri plates upside down?

to prevent contamination and water condensation from damaging culture

Temperature used for incubation?

37 C

Why is TSA known as general purpose medium?

it supports the growth of a wide variety of microorganisms

What is a colony? What does a colony represent?

a visible cluster of microorganisms growing on a solid medium originating from a single parent cell

Characteristics of bacterial colonies

shape, margin, elevation, size, texture, appearance, pigmentation, optical property

ocular lens (eyepiece)

what is 9

revolving nose piece

what is 2

objective lens

whats 4 / 5

condenser

whats 7

the stage

whats 11

fine focus and coarse focus knob

whats 13 and 12

Purpose of immersion oil

goes between slide and lens and prevents light from bending and illuminates the image

whats total magnification

ocular lens (10x) times objective lens

ex. 10x x 40x = 100x

What characteristic of a microscope enables one to switch from one objective to another without altering the focus?

parfocality

identify

identify erythrocytes

identify

identify lymphocytes

identify

identify neutrophils

identify

identify platelets

identify

identify protozoa

identify

identify yeast

identify

identify taenia solium

identify

identify staphylococci

identify

identify streptococci

identify

identify diplobacilli

identify

identify streptobacilli

Aseptic technique and how

prevent contamination of our culture, media (sterile) and environment

-using sterile media and tools (inoculating loop)

-using Bunsen burner

-PPE

-washing hands

-disinfecting work area

pure culture vs mixed culture

- only one species of microbe growing on plate/ broth

- 2 or more species growing together on plate/ broth

The most common method to obtain a pure culture of a bacterium

streak plate (using the T Streak method)

Why is it important to only work with isolated colonies when performing experiments in microbiology?

allows a specific microorganism to be studied

differential stain purpose

reveals differences in cell structure or composition

What color would a Gram negative cell be after the decolorization step?

colorless

What color would a Gram positive cell be after the decolorization step?

purple

Why do you need to blot the slide dry before decolorizing?

prevents dilution and allows to tell difference between Gram +/-

What do you think would happen if you forgot to heat fix the cells during gram staining

bacteria would wash off the slide during staining

What color would a Gram positive cell be if you stained fist with safranin and counterstained with crystal violet?

still purple

simple stain vs differential stain

simple only uses one dye and differential is used to tell difference between microbe types (gram +/-)

Gram Staining procedure

step 1. flood heat fixed slide with Crystal Violet

step 2. flood slide with Iodine to bind to the Crystal Violet

step 3. apply decolorizer (Ethanol/ Acetone) to differentiate between types

step 4. Counter stain pink with Safranin (stains gram- pink and gram+ remains purple)

how would the gram +/- look if you forgot to heat fix

there would be no visible cells

how would the gram +/- look if you forgot crystal violet?

gram + : pink

gram - : pink

how would the gram +/- look if you forgot Iodine?

gram + : pink

gram - : pink

how would the gram +/- look if you forgot decolorizer?

gram + : purple

gram - : purple

how would the gram +/- look if you forgot safranin?

gram + : purple

gram - : no color

method used in lab to look for bacteria motility?

hanging drop

what is selective media

'selects' certain microbes to grow while inhibiting others (MacConkeys Agar and Mannitol Salt Agar)

what is differential media

uses indicators to tell the difference between certain species of bacteria

differential media vs differential stain

the media differentiates bacteria based on the growth on the plate, while the stain differentiates bacteria based on their appearance under a microscope

what makes MAC selective & differential

- Crystal violet and bile salts

- Lactose and a pH indicator Neutral Red

what makes MSA selective & differential

selective: high (7.5%) NaCl concentration

differential: mannitol and a pH indicator Phenol Red

What would growth on a MAC plate indicate (with genus and species name)

gram -

Escherichia coli

What would growth on a MAC plate that is pink indicate (with genus and species name)

- Gram-

-lactose fermenter

-Escherichia Coli

What would growth on a MAC plate that is colorless indicate (with genus and species name)

- gram-

-Non Lactose fermenter

-Salmonella Enterica

what would no growth on a MAC plate indicate (with genus and species name)

- Gram +

-Staphylococcus Aureus

What would growth on a MSA plate indicate (with genus and species name)

- Gram+

- Staphylococcus Aureus

- halotolerant

What would growth on a MSA plate that is yellow indicate (with genus and species name)

-Gram +

-Mannitol Fermenter

-Staphylococcus Auerus

-halotolerant

What would growth on a MSA plate that is colorless indicate (with genus and species name)

-Gram +

-Non Mannitol fermenter

-Streptococcus Epidermis

-halotolerant

What would no growth on a MSA plate indicate (with genus and species name)

-Gram -

-Escherichia Coli

What makes blood agar plate differential

detects microorganisms different abilities to produce hemolysins that lyse RBC

-uses 5% sheep's blood

Beta hemolysis

-appears clear zone around a colony

-complete lyses of RBC

-Staphylococcus Aureus & Streptococcus Pyogenes

Alpha hemolysis

-appears greenish zone around colony

-partial lyses of RBC

-streptococcus pneumoniae

gamma hemolysis

-no visible change in agar around the colony

-no breakdown of RBC

-staphylococci / streptococci

What is being tested in the phenol red broths

ferments a carbohydrate and produces acid, a differential test

What 4 sugars do the phenol red broths test for?

glucose, sucrose, lactose, mannitol

what does red broth with growth, no gas bubble indicate

neutral reaction (-) and pH at 7

what does yellow broth with no gas bubble indicate

acid produced (+) and lower pH

what does yellow broth and gas bubble in Durham's tube indicate

acid and gas produced (+/g)

Clinically, what does the catalase test determine?

if the microorganism produces the enzyme catalase

What is the substrate/reagent used for the catalase test?

hydrogen peroxide

what does it mean to be catalase positive

-produces the enzyme catalase

-aerobe / facultative anaerobe

-staphylococcus epidermis

-foams bubbles of O2

what does it mean to be catalase negative

-anaerobic / facultative anaerobe

-*streptococcus pyogenes*

-doesn't foam

what does the oxidase test determine

if a microorganism produces cytochrome c oxidase that is in the electron transport system

The _______ test is used to determine the presence/absence of the respiratory enzyme _________ in bacterial species.

the OXIDASE test is used to determine the presence/ absence of the respiratory enzyme cytochrome oxidase in Gram - bacteria

What does a positive oxidase reaction look like?

should become dark purple within 30 seconds**

What does a negative oxidase reaction look like?

doesn't change color or takes longer than 30 seconds to become dark purple

examples of oxidase positive bacteria

gram- pseudomonas and vibrios ((Pseudomonas fluorescens)

what does the urea test test for

for presence of urease, uses a pH indicator

How is the presence of the enzyme urease detected in the medium?

when the bacterium hydrolyzes urea, ammonia is released and increases pH, causing indicator to change to a magenta color indicating a pos test

What is the substrate and product for the enzyme urease?

substrate: urea

products: carbon dioxide and ammonia

Name a bacterial Genus/organism that would test positive for urease?

Staph. saprophyticus

gelatin hydrolysis test purpose

used to determine the ability of an organism to produce extracellular proteolytic enzymes (gelatinases) that can liquefy gelatin in the vertebrate connective tissue.

What is a positive test for gelatinase and how do we interpret it?

a gelatinase positive organism will secrete gelatinase when in gelatin, and it will liquefy the medium (takes 7-14 days of incubation)

What is a negative test for gelatinase and how do we interpret it?

the organism will not liquefy the medium

Name a bacterial Genus/organism that would test positive for the gelatin hydrolysis test

Pseudomonas fluorescens

What information can be gathered from looking at a gram stained bacterial smear?

Gram reaction, Cell Size, Cell shape, Cell grouping