Micobiology Lab Quiz 2

Differential stain

• uses more than one stain

• Allows you to differentiate between different types of bacteria

Gram stain

• based on bacterial cell wall properties

• Differentiates between Gram-positive and Gram-negative bacteria

Peptidoglycan

Composes the bacterial cell wall

Peptides and sugars/carbohyrdates

Composes peptidoglycan

Carbohydrate backbone

Consists of repeating sugars

NAG and NAM

Repeating sugars of the carbohydrate backbone

Glycosidic bonds

Bonds that link NAG and NAM

Peptide bonds

Bonds that link carbohydrate backbones

Gram-positive bacteria

• thick layer of peptidoglycan

• Contains teichoic acid

• No outer membrane

Wall teichoic acid

Connects peptidoglycan layers to each other

Iipoteichoic acid

Connects peptidoglycan to lipids in plasma membrane

Gram-negative bacteria

• thin layer of peptidoglycan

• No teichoic acid

• Contains an outer membrane

Outer membrane composition

• phospholipid bilayer

• Porins

• Lipoplysaccharide (LPS)

Porins

Channels that allow substances to enter /exit

Lipopolysaccharide (LPS)

Extends outward from phospholipid bilayer

Lipoproteins

Anchor outer membrane to cell wall

Crystal violet

• Primary stain for Gram Stain ( ~2 min )

• Secondary stain for Capsule Differential Stain

• Stains purple

Gram’s Iodine

Mordant (binding agent) in Gram Stain (~2 min)

Acetone Alcohol

Detaining agent (destainer) in Gram Stain (~3-8secs)

Safranin

Secondary stain (counter stain) in Gram Stain and Endospore Differential Stain (~2min)

Purple

Gram positive stain

Pink

Gram Negative stain

Destain for too long

Gram-positive bacteria appear falsely gram-negative

Destain for too short

Gram-negative bacteria appear falsely gram-positive

Cells are too old

Cell wall can degrade or change

Capsule

gelatinous coat surrounding some bacteria

Uncharged polysaccharides

Composition of capsule

Functions of the capsule

• increases bacterial virulence

• Prevents desiccation

• Adheres to surfaces

• Resists phagocytosis

• Nutritional reserve

Virulence

Degree of disease a pathogen can cause

Desiccation

Drying out

Capsule differential stain

Identify bacteria with capsule

Nigrosin

Negative stain in capsule differential stain (stains background black)

Acid-fast bacteria

Cell wall contains mycolic acid

Mycolic acid

Waxy lipid that makes cell impermeable to most stains

Functions of mycolic acid

Prevents desiccation and digestions after pathogenesis

Mycobacterium and Nocardia

Acid-fast genera

Mycobacterium tuberculosis

Tuberculosis

Mycobacterium leprae

Leprosy

Carbolfuchsin

• Primary dye in Acid-Fast Stain

• Contains phenol

• Dark red basic dye

• Must be added over steam

Acid Alcohol

destainer in Acid-Fast stain

Methylene Blue

Counterstain in Acid-Fast Stain

Blue

Non-acid fast bacteria stain color

Red

• Acid-fast bacteria stain color

• Vegetative body stain color

Vegetative body

Actively metabolizing

Endospore

Not actively metabolizing (only produced by certain bacteria)

Bacillus and Clostridum

bacteria types that produce endospores

Bacillus anthracis

Anthrax

Clostridium botulinum

Botulism

Clostridium tetanus

Tetanus

Clostridium difficile

Colitis

Sporogenesis

Process that endospores are formed through

Harsh environments

What causes sporogenesis?

One endospore

One vegetative body forms:

Endospore structure

Smaller and more compact that vegetative bodies

Endospore coat

Thick layer of keratin + calcium diplocholinate

Functions of endospore structure

• resistant to heat, alcohol, UV, drying, etc.

• Can remain dormant for long periods of time

Endospore germination

• occurs when environment improves

• One Endospore—> one vegetative body

• NOT a reproductive process

Endospore differential stain

Differentiate between vegetative cells and endospores

Malachite green

• primary stain in Endospore differential stain

• Added over heat

  • Allows stain to penetrate thick endospore coat

Water

Detainer of endospore differential stain

Green

Endospore stain color

Aerobe

Utilize O2

Obligate aerobe

Require O2

Microaerophiles

Prefer low O2 and high CO2

Anaerobes

Do not utilize oxygen

Obligate anaerobe

Cannot tolerate the presence of O2

Aerotolerant

Cannot utilize O2, but tolerate it fairly well

Facultative anaerobe

Capable of living with or without O2, but prefer it

Sodium thioglycollate

Reducing agent that bonds to some of the O2 —> H2O

Small amount of agar

Reduces the diffusion of O2 from the air into medium

Resazurin

O2 indicator (changes color depending on if O2 is present or not)

O2 present

Resazurin turns pink

O2 absent

Resazurin is colorless

Anaerobic Pouch

Creates anaerobic environment

Gas Pak

• absorbs O2

• Produces CO2

Blue

Methylene Blue color when O2 is present

White

Methylene Blue color when O2 is absent

Candle Jar

Add lit candle to a jar

Candle consumes most of the O2 and generates CO2

Creates microaerophilic environment

Motility

Self-directed movement of an organism via cilia, pseudopods, or flagella

Cilia

Short-hair like processes (eukaryotic)

Pseudopods

“Fake foot” projection of cytoplasm (eukaryotic)

Spirillum volutans

Flagellated bacteria example

Flagella

Longer whip-like appendages (prokaryotic)

Brownian motion

Vibrational movement caused by collision with H2O molecules

Monotrichous

One single flagellum

Amphitrichous

Flagella on both ends

Peritrichous

Flagella surround entire cell

Lophotrichous

Multiple flagella at one end

Ways to check for motility

• wet mount (microscopic)

• Hanging drop (microscopic)

• Semi-solid agar (culture)

0.5% agar

Agar percent for semi-solid agar

1% agar

Normal solid media agar %

Non-motile

Crisp line of growth

Motile organism

Hazy growth away from stab line

Only accounts for living organisms

Advantage of semi-solid agar test

Disadvantages of semi-solid agar motility test

• requires overnight incubation

• Results can be difficult to interpret

Heterotrophic

Must get carbon form other sources

Saprophytic

Can obtain nutrients from dead, organic matter

Mycosis

Fungal infection

Chitin

Cell wall of fungi

Low pH and high sugar

Preferred environment of fungi