Micro Lab Midterm (exercises 1-9)

What do you clean the bench with before lab?

70% EtOH

what do u do if u spill something?

do not try to move it or clean it on ur own!! let a TA and the ppl around u know immediately

what is proper lab attire?

• wearing provided lab coat entire time until u leave. lab coats can never leave lab

• gloves and goggles worn at all times

• long pants down to ankles and closed-toe shoes

• long hair tied back

What do you clean the bench with after lab?

Bleach wipes

Where do you label Petri dishes?

Bottom of the plate where medium is. label around the edge for easy observation of growth on plate

• label ur seat #, date, and organism u inoculated

How do you store Petri dishes?

Inverted (bottom on top) → this is so condensation that might form on the lid doesnt fall into the plate and ruin ur inoculation

What temperature are Petri dishes incubated at?

37 degrees Celsius

What is used to label test tubes?

China markers

What is the thing called that sterilizes the inoculating loop and openings of test tubes?

Microincinerator. only touch the base

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

¾ make sure to not rest it against the inside of the incinerator

what types of media preparations do we handle?

general purpose, selective, differential

• broth

• agar slants

• agar deep tube

• agar plate

Asepsis means

without infection

how do u handle and label test tubes?

never carry by the cap. transport them in the tube rack and hold them in ur non-dominant hand

• never put caps down on benchtop → remove cap w ur dominant hand and hold it throughout transfer

• label tubes on clear portion, never on the cap. use china marker

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

Resuspend if broth culture

1) sterilize loop

2) uncap tubes (avoid touching cap opening w glove)

3) sterilize tubes

4) transfer from culture to fresh medium

5) sterilize tubes

6) recap tubes

7) 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 dyes)

  • positively charged chromophores

  • ex: Crystal Violet or Methylene Blue

• Anionic (acidic dyes)

  • negative ion exhibits color

  • ex: Acid Fuchsin, Congo Red, Nigrosin

Cationic dyes are

Basic, positively charged chromophores

• positive ion exhibits color

• ex: Methylene Blue or Crystal Violet

actually stains the cell → attracted to net negative charge of 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 very good for examining morphology and size

  • no heat = distortion of bacterial cells minimized

negative stain

a anionic dye that doesnt penetrate the cell

• repelled by negative charge at cell surface → we stain the background instead so u see transparent cells against colored background

• v good to examine size and morphology of cells cuz we do not heat fix! so distortion of bacterial cells is minimized

• good for organisms that dont stain easily

• ex: Nigrosin (black anionic dye)

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

simple stains

use cationic dye to stain all cells present

• good to distinguish diff morphologies, arrangements, and relative sizes. will have some distortion cuz we need to heat fix

• colors the cells and provides contrast when using bright field microscopy

• positive charge of the dye interacts w negative charge on bacterial cell wall → colors cell surface

ex: Crystal Violet

Are simple stains cationic or anionic?

Cationic ( + charge will interact w - cell charge to actually stain the surface of cell)

how many dyes are used in a simple stain?

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 of shape + morphology

how do u smear prep from broth?

1) transfer 1 loopful of culture to clean slide and spread for max thinnness

2) air dry

3) heat fix by holding over microincinerator for 10 seconds - use clothespin!

how do u smear prep from slant or plate?

these r solid media so:

1) add 1 loopfull of water to slide

2) grab bacterial sample by softly tapping loop in media and then mixing it with water on slide to spread

3) allow to air dry

4) heat fix by holding over microincinerator for 10 seconds - use clothespin!

How do you carry the microscope?

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

how do u properly store microscope?

• clean oil off 100x using lens cleaner and kimtech wipe

• lower stage as low as possible

• move 4x objective into position

• make sure cord is wrapped up

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 (4x, 40x, 100x oil)

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

used for 4x objective

What is the focus called that is smaller?

Fine focus → moves stage up and down

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

what r the 3 principles of microscopy?

1) illumination

• light source and condensor to direct light towards obj. lens.

• iris diaphragm to adjust diameter of cone of light

2) magnification

• ocular = 10x

• Objectives = 4x, 10x, 40x, 100x

• total magnification = ocular x objective

3) resolution

• the smallest distance btwn 2 objects which can be seen as separate

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

• cuz oil has same refractive index as glass and oil will stop the refraction of light btwn specimen and lens

• oil must be touching lens.

doesnt help w magnification but helps w light!!

How do you calculate resolution?

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

• d = resolution

• lambda = wavelength of light (nm)

• NA = numerical aperture (light gathering capacity of lens)

smaller d = better resolution

What does d stand for?

Resolution

What does NA stand for?

Numerical aperture (light gathering capacity of the lens)

What does lambda stand for?

Wavelength of light (nm)

How do you improve d (resolution)?

Smaller d = better resolution (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

• ex: Corynebacterium diptheriae is club-shaped

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

• 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

• increases how well crystal violet binds to cell wall

What is the decolorizer/differential step in gram staining?

95% ethanol

What is the secondary dye/counterstain in gram staining?

Safranin

what would happen if we skip the 95% ethanol in gram staining?

all cells would look purple!

• after 95% ethanol, gram (-) cells become clear and gram (+) cells remain purple. then Safranin secondary dye stains gram (-) cells pink

gram stain process

1) crystal violet - 1 min

2) grams iodine - 1 min

3) 95% ethanol - 10 secs

4) safranin - 1 min

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

fastness

whether or not a organism is going to retain the primary dye when challenged by a decolorizer

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