Micro Lab Midterm

What do you clean the bench with before lab?

70% EtOH

What do you clean the bench with after lab?

Bleach wipes

Where do you label Petri dishes?

Bottom of the plate where medium is

How do you store Petri dishes?

Inverted (bottom on top)

What temperature are Petri dishes incubated at?

37oC

What is used to label test tubes?

China markers

What is the thing called that sterilizes the inoculating loop?

Microincinerator

How much of the loop needs to be glowing red hot for sterilization?

3/4

Asepsis means

without infection

What are the 7 (8) steps of a two-tube transfer?

Resuspend, sterilize loop, uncap tubes, sterilize tubes, transfer from culture to fresh medium, sterilize tubes, recap tubes, sterilize loop

How do you obtain culture from a broth tube?

Stab

• liquid

How do you obtain culture from a slant tube?

Fishtail

Staining provides

contrast to identify morphology, size, and arrangement of bacterial sample

Staining is useful for identifying

morphology, shape, and arrangement

What are the different types of staining?

Simple (cationic), differential, structural, negative

What are the types of dyes used in staining?

• Cationic (basic)

• Anionic (acidic)

Cationic dyes are

Basic, positively charged chromophores

• positive ion exhibits color

• ex: Methylene Blue or Crystal Violet

actually stains the cell

Examples of cationic dyes

Methylene blue and crystal violet

Anionic dyes are

Acidic, negatively charged

• negative ion exhibits color

• ex: Acid fuchsin, congo red, Nigrosin

doesn’t penetrate cell, cell is not stained → background takes color

Examples of anionic dyes

Acid fuchsin, Congo red, nigrosin

A negative stain doesn't

penetrate cell, stains background

• negatively charged stain repels negative cell

Are negative stains cationic or anionic?

Anionic

Are negative stains heat fixed?

No

• cuz of this its good for examining morphology and size

  • no heat = distortion of bacterial cells minimized

Negative stains are useful for organisms that

don't stain easily

• it doesnt actually stain cell so it doesnt matter if it doesnt stain easily

Example of a negative stain dye

Nigrosin (black anionic dye)

How does a negative dye work?

Repelled by negatively charged cell surface

Are simple stains cationic or anionic?

Cationic

Simple stain dyes

one dye used to stain all cells present

What dye do we use for simple stains?

Crystal violet

How does a simple stain work?

Positive charge interacts with negative charge of bacterial cell wall to color cell surface

Are simple stained slides heat fixed? What does this cause if so?

Yes, some distortion

How do you carry the microscope?

With two hands - one holds the handle and other supports base

What is the part of the microscope called that you look through?

Ocular

What are the parts of the microscope called that allow you to see the microbe?

Objectives

What is the part of the microscope called that holds your slide?

Stage

What is the focus called that is bigger?

Coarse

• moves stage up/down

What is the focus called that is smaller?

Fine focus

What adjusts the diameter of the cone of light so that is just fills the objective lens?

Iris diaphragm

What is the smallest distance between two objects which can be seen as separate?

resolution

How do you calculate magnification?

Ocular (10) x objective = total magnification

• 10x (ocular) x 100x (objective) = 1000x magnification

Which objective is the oil immersion?

100X

• for both cationic and anionic stains

How do you calculate resolution?

d = lambda (wavelength of light nm)/(2NA)

What does d stand for?

Resolution

What does NA stand for?

Numerical aperture (light gathering capacity of the lens)

What does gamma stand for?

Wavelength of light

How do you improve d (resolution)?

Smaller d = better (increase NA or decrease gamma)

Streptococci

Chains of cocci

Sarcina

tetrads (square/4 cocci)

• 4 in front and 4 in back

Staphylococci

grape-like clusters of cocci

what shape are bacilli?

rod shaped

• strepto = chain

• diplo = pairs

Vibrio cholerae are what shaped?

Comma shaped

Spirillum volutans are what shaped?

Rigid and spiral

Pleomorphic

Morphology that is not easily categorized

Which stain uses one dye to stain all cells present?

Simple

Which stain detects differences between organisms through the use of many dyes and reagents?

Differential stain/gram stain

Who developed the gram stain?

Hans Christian Gram

Why did Hans Gram develop the gram stain?

He was trying to differentiate bacteria causing pneumonia from the lung tissue of deceased patients

Gram +

Thick petidoglycan layer

Gram -

Thin peptidoglycan layer, 2 membranes

What is the primary dye in gram staining?

Crystal violet

What is the mordant in gram staining?

Gram's iodine

What is the decolorizer in gram staining?

95% ethanol

What is the secondary dye/counterstain in gram staining?

Safranin

What color does gram + stain?

Purple

What color does gram - stain?

Red

What types of staining are differential?

Gram stain and acid-fast stain (Ziehl-Neelsen method)

What types of staining are structural?

Endospore stain (Schaeffer-Fulton Method)

Acid fast staining is also called

Ziehl-Neelsen

Acid-fast microbes have what in their cell walls that makes them hard to stain? Why?

Mycolic acid, waxy and resists

What do we do to force the primary dye to penetrate the waxy cell wall in acid-fast staining?

Use steam

What means to retain the dye when challenged by a decolorizer?

Fastness

What is the primary dye in acid-fast staining?

Carbol fuchsin

• bright pinky/red

What is the decolorizer in acid-fast staining?

Acid alcohol

What is the secondary dye/counterstain in acid-fast staining?

Methylene blue

Procedure for acid-fast staining

• Heat fixed slide

• Steam with carbon fuchsin for 3 minutes and rinse with water

  • need to steam to penetrate waxy cell wall in microbes w/ high mycolic acid content

• Decolorize with acid alcohol for about 12 seconds

• Counterstain with methylene blue for 1 minute

Pink/violet = acid-fast

Blue = acid-fast negative

Acid fast cells stain

Cerise/pink/violet

Non-acid fast cells stain

Blue

What is an example of a microbe that is acid fast?

Mycobacterium

• ex: M. tuberculosis and M. leprae

• have high mycolic acid content

What is the causative agent of Tuberculosis?

Mycobacterium tuberculosis

• acid-fast

What is the causative agent of leprosy/Hansen's disease?

Mycobacterium leprae

• acid-fast

Spore staining is also called

Schaeffer-Fulton method

Endospores release structures called

free spores

How do you force the primary dye into resistant endospores?

Steaming

• cuz spores r highly resistant

What is the primary dye in spore staining?

Malachite green

What is the decolorizer in spore staining?

Water

What is the secondary dye/counterstain in spore staining?

Safranin (reddish)

What color do spores and cells stain?

Spores stain green

cells stain pink

spores

produced by some organisms in response to poor environmental conditions. They are a dormant form of bacteria “doomsday bacteria”.

• if they release spore its a free spore

• if spore is still inside bacteria then its an endospore

• if bacteria is yet to produce spores then its a vegetative cell

What color do spores stain?

Green

Spore staining procedure

• Heat fixed slide

• Stain and steam with malachite green for 3 minutes

• decolorize with water very well

• Counterstain with safranin

Spores = green

pink = no spores or vegetative cells

What are cells that have yet to produce spores called?

Vegetative cells

What are cells with spores called?

Endospores (green spore inside pink bacterial cell)

What are some spore forming bacteria?

Bacillus

• B. anthracis (causes anthrax)

Clostridium

• C. botulinum (causes botulism and used for Botox)

• C. tetani (causes tetanus)

• C. difficile (causes colitis from too many antibiotics throwing off gut microbiome)

BOTH are gram positive rods!!

Pure culture contains

a single microbial species

What does a pure culture aim to do?

Dilute bacterial samples in order to separate individual cells that can grow into isolated colonies when plated

When is general purpose media used?

When trying to isolate microbes from mixed culture

What is often preferred when trying to obtain pure cultures?

Solid media/agar

• microvbes can’t metabolize/breakdown agar

What is a solidifying agent derived from seaweed?

Agar

Is a pour plate used quantitatively or non-quantitatively?

Both depending on method

In the pour plate method, a culture is diluted in what and at what temp?

Molton agar (series of tubes) more than 40 degrees