Microbiology Lab Midterm

Compound microscope

1. Ocular-eyepiece lens (usually 10x)

2. Objective-nosepiece lenses (commonly 4x, 10x, 45x, and 100x)

At what magnification do you use the oil immersion technique?

100x.

Total Magnification =

Magnification (Objective) x Magnification (Ocular)

Important Parts of the Microscope

Framework, Stage, and Lens System

Framework

Arm and base are the structural parts of the microscope which support the basic frame.

Stage

Holds the slide. The mechanical stage clamps the slide and moves the slide around the stage.

Lens System

Oculars, Objective, and Condenser

Oculars

Eyepiece lenses (usually 10x magnification)

Objectives

Lenses attached to rotatable nosepiece, common magnification of 4x, 10x, 45x and 100x.

Parfocalized microscope focusing adjustments are not to be made when...

changing the objective lenses.

The oil has approximately the same refractive index as glass to prevent...

light loss due to diffraction (bending of light rays) which would occur if light traveled from one refractive index to another (eg. glass to air)

As magnification of the objective lens increases...

the working distance decreases.

Working distance

distance between the object on slide and the objective lens, when in focus.

Condenser

directs light towards the objective lens in bright field microscopy.

In dark field microscopy, the condenser...

directs light at oblique angles away from the objective lens in a manner that allows only objects in the field of view to redirect or scatter light into the objective lens. This causes objects to appear white on a dark field.

Iris Diaphragm

lever located in the condenser that adjusts the diameter of the cone of light so that it just fills the objective lens.

As you close down the Iris Diaphragm...

1. The light intensity decreases

2. Contrast improves

3. Depth of field increases

4. Limit Resolution (with oil immersion lens)

Resolution

Resolving Power. Expressed as d.

d = the smallest distance between two objects which can be seen as separate

d = the diameter of the smallest resolvable object

d = wavelength / 2 NA

NA = numerical aperture

To improve resolution...

lower d.

d can be decreased by...

lowering wavelength or increasing the NA.

Cocci

More or less spherical organisms.

Bacilli

Cylindrical organisms.

Helicoidal

Spiral shaped organisms.

Incompletely separated cocci may appear in a number of different patterns depending upon...

the plane in which they divide and how they remain attached.

Diplococci

pairs. Divide in one plane.

Streptococci

chains. Divide in one plane.

Tetracocci

tetrads. Divide in two planes.

Staphylococci

clusters. Divide in three planes irregularly.

Sarcinae

cuboidal packets. Divide in three planes regularly.

Coccobacillus

very short and almost appears spherical, but they are just slightly longer in one direction than the other.

Fusiform Bacilli

tapered at the ends, appearing as football like in shape.

Filamentous Bacillary Forms

grow in long threads.

Most microorganisms are difficult to see using light microscopy due to...

their size and the lack of contrast between the cell and the environment.

The contrast is improved with...

the help of dyes.

Dyes are...

organic compounds containing a chromophore with affinity for cellular material.

Types of dyes

1. Cationic (basic dyes, positively charged chromophore) - Methylene Blue, Crystal Violet

2. Anionic (acidic dyes, negatively charged chromophore) - Acid fuschin, Congo Red, Nigrosin

3. Fat Soluble (no charge): Sudan Black stains granules of Poly-B-OH-butyric acid

4. Insoluble Dyes (water insoluble): India Ink (colloid suspension of carbon particles)

Negative Staining

Stains background, not the cell in brightfield microscopy (Not Dark field Microscopy)

Types of dyes used in Negative Staining

1. Nigrosin - a black anionic (negatively) charged dye. The negatively charged dye is repelled by the negatively charged surface of the bacterial cell.

2. India Ink - an insoluble dye (a colloidal suspension of carbon particles) which does not penetrate the cell surface.

Simple Staining

One dye used to stain all cells the same color. Can be used to tell morphology (shape) and size [although negative staining is better for size]. Cationic dyes are positively charged and are attracted by ionic forces to the negatively charged surface of the bacterial cell. Common dyes are methylene blue and crystal violet.

Differential Staining

Staining procedure which causes cells to stain differently based on properties of the cell.

Gram stain

a differential stain procedure that causes cells to stain differently based on characteristics of their cell wall. Cells are stained with crystal violet, then fixed with iodine forming a crystal violet-iodine complex within the cell. Ethanol is then added as a decolorizer. The gram neg. cells are then counterstained with saffranin.

Gram-positive microorganisms have a higher ____________ and lower ______ content than gram-negative microorganisms.

1. peptidoglycan

2. lipid

Why are gram negative cells easily decolorized?

because the ethanol dissolves the high lipid cell wall allowing the crystal violet-iodine complex to readily exit the cell.

How do gram positive cells resist colorization?

due to the difference in cell wall consistency retaining the crystal violet-iodine complex.

What color will the gram positive cells be after the primary stain (crystal violet)?

purple.

What color will the gram negative cells be after the primary stain (crystal violet)?

purple.

What color will the gram positive cells be after the mordant (iodine) is added?

purple.

What color will the gram positive cells be after the decolorizer (ethanol) is added?

purple.

What color will the gram positive cells be after the counterstain (saffranin)?

purple.

What color will the gram negative cells be after the mordant (iodine) is added?

purple.

What color will the gram negative cells be after the decolorizer (ethanol) is added?

colorless.

What color will the gram negative cells be after the counterstain (saffranin)?

pink.

Acid Fast Stain

a differential stain procedure that causes cells to stain differently based on characteristics of their cell wall. Cells are steamed in the presence of calbol fuschin and decolorized with acid alcohol. Cells which are "acid fast" (microorganisms have a high wax content in their walls) will not decolorize and remain red, while non acid fast organism will readily lose their stain and become colorless. These cells are then counterstained with Methylene blue.

Acid Fast microorganisms have a high...

wax content in their walls, which requires the use of steam to allow dye to penetrate the cell.

What color will the Acid Fast positive cells be after the primary stain (Carbol Fuschin)?

red.

What color will the Acid Fast positive cells be after the decolorizer (acid alcohol) is added?

red.

What color will the Acid Fast positive cells be after the counterstain (Methylene Blue)?

red.

What color will the Acid Fast negative cells be after the primary stain (Carbol Fuschin)?

red.

What color will the Acid Fast negative cells be after the decolorizer (acid alcohol) is added?

colorless

What color will the Acid Fast negative cells be after the counterstain (Methylene Blue)?

blue.

Two genera of Acid Fast Organisms (All other genera are Non-Acid Fast)

Mycobacterium and Nocardia.

Mycobacterium do not gram stain well...

if mature, because of high wax content within walls; if young, appear as gram + tapered rods that sometimes fragment.

What are the two important species of Mycobacterium?

tuberculosis and leprae.

Spore Staning

Some microorganisms produce heat and chemical resistant structured called endospores or free spores. To stain the spores the cells must be steamed to allow for the dye (malachite green) to enter the spores. Once the spores are stained, all other microorganisms and vegetative cells can easily be decolorized with water, while the free spores and endospores retain the malachite green. The other microorganisms and vegetative cells are then counterstained with Safranin.

Endospores appear as...

a green center within a pink sporangium.

Free Spores appear as...

small green oval bodies.

Three genera of Spore forming organisms

Bacillus - Aerobic, gram + rod

Clostridium - Anerobic, gram + rod

Sporsarcinae - Cocci

Anerobic green

endospore/free spores Clostridium

Aerobic green

endospore/free spores of Bacillus

Anerobic pink

vegetative/sporangia of Clostridium

Aerobic pink

vegetative/sporangia of Bacillus

What color will a spore be after the primary stain (Malachite Green)?

green

What color will a spore be after the steam?

green

What color will a spore be after the decolorizer (water)?

green

What color will a spore be after the counterstain (Safranin)?

green

What color will a non spore be after the primary stain (Malachite Green)?

green.

What color will a non spore be after the steam?

green.

What color will a non spore be after the decolorizer (water)?

colorless.

What color will a non spore be after the counterstain (Safranin)?

pink.

Five Methods of Tube Media Preparation

1. Pour = 15 - 20 mL of liquid agar used to pour into a plate

2. Broth = 5 - 7 mL of liquid media

3. Deep = 5 - 7 mL of media which has solidified in an upright position

4. Slant = 5 - 7 mL of media which has solidified at an angled position

5. Fermentation Broth = Broth with Durham Tube added

Natural Media

Media composed of complex raw materials whose actual chemical composition is unknown.

What is an example of Natural Media?

Nutrient Agar

Synthetic Media

Media whose exact chemical composition is known and in many instances is designed for isolation, selection or differentiation of specific types of microorganisms.

Selective Media

a media which favors the growth of one type of microorganism over another. This is accomplished by either inhibiting unwanted microorganisms or enriching - providing conditions which are preferential to the desired microorganism.

Differential Media

a media which differentiates or distinguishes between different types of microorganisms based on differences in appearance of growth or color changes.

Selective and/or Differential Media

Phenylethyl Alcohol Agar (PEA), Desoxycholate Agar (DES), Eosine Methylene Blue (EMB), and Blood Agar.

Phenylethyl Alcohol Agar (PEA)

Selects for the growth of gram positive microorganisms, because Phenylethyl Alcohol is inhibitory to the growth of gram negative organisms

Desoxycholate Agar (DES)

Selects for gram negative microorganism, because Desoxycholate Agar is inhibitor towards the growth of gram positive organisms. Differentiates for lactose fermentors (lactose + microorganisms from lactose negative). Lactose fermentors produce acid which precipitates the bile salts in the media and absorbs the neutral red dye, therefore appearing Red. Non-fermentors do not do this and do not appear red.

Eosine Methylene Blue (EMB)

Selects for gram neg. organisms.

Differentiates lactose +/- microorganisms. Lactose + show a color change, Lactose - do not show a color change

Can further differentiate Lactose + fermentors based on the amounts of acid produced during lactose fermentation.

Mixed Acid Fermentors produce more acid and produce colonies with dark blue-black centers (center is almost the size of the whole colony) and some microorganisms like Escherichia coli, produce a metallic green sheen

Butanediol Fermentors produce less acid so that the colonies have pale pink to lavender centers. The centers are only a small part of the colony (ie bull's-eye colonies), and will not have a metallic green sheen like Enterobacter.

Blood Aga

Differentiates microorganisms based on their reactions on blood.

Gamma Hemolysis

No blood hemolysis, no zone of clearing around the colony.

Beta Hemolysis

Complete blood hemolysis and complete clearing around the colony.

Alpha Hemolysis

Partial blood hemolysis and partial clearing around colony. Partial clearing sometimes appears green due to partial reduction of hemoglobin in blood.

Biochemical Tests

Tests used to determine physiological characteristics of microorganism, particularly in terms of bacterial enzymes and the chemistry of biooxidation.

Starch Agar

Tests for the presence of Amylase, which hydrolyses starch to simple sugars. Iodine is added to starch plate and appears blue/black when interacting with starch. If amylase is present starch will be hydrolyzed and the blue/black color will not be observed around the amylase positive colonies.

Milk Agar

Tests for the presence of the enzyme Caseinase, which hydrolyzes casein (a predominant protein in milk) into amino acid products. Casein gives milk its white color so a breakdown in casein causes the milk plate to lose its white color and become clear around the Caseinase positive colonies.

Lipase Plate

Tests for the presence of the enzyme lipase which hydrolyzes fat to form glycerol and fatty acids. The production of the fatty acids lowers the pH just enough to produce a dark blue precipitate when a microorganism is Lipase positive.

Sugar Fermentation Tubes

Used to determine if a microorganism can ferment particular sugars. The fermentation tubes contain the sugar of interest (glucose, lactose, mannitol), pH indicator (phenol red) and a Durham tube. If a microorganism is able to ferment the sugar being tested the result of the fermentation will result in the production of acid, therefore lowering the pH of the solution and causing the liquid to turn yellow from its original red color. Some microorganisms also produce gas during fermentation, which is important to know when identifying unknown bacteria. This gas will collect in the Durham tube and appear as a void or bubble in the inverted tube. An alkaline reaction can also occur, which is due to the utilization of the peptone in the broth and not the testing sugar. An alkaline reaction is indicated by the darkening of the red pH indicator color.

In sugar fermentation, yellow equals...

acid.

In sugar fermentation, red to dark red equals...

negative or alkaline.