Microscope Basics:

Simple Microscope: One lensCompound Microscope: Two lenses

1. Ocular-eyepiece lens (usually 10x)2. Objective-Nosepiece lenses (commonly 4x 10x 45x 100x (oil immersion))

Mag(Total) = Mag(objective) x Mag(ocular) Total=Objective * Ocular

Important Parts of the Microscope: Framework - Arm, Base 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, Objectives, and Condenser

o Oculars – eyepiece lenses (usually 10x magnification)o Objectives – lenses attached to rotatable nosepiece common magnifications of 4x, 10x,

45x (low power, high-dry objectives) and 100x (oil immersion lens). Parfocalized microscope focusing adjustments do not have to be made when

changing objective lenses

Oil immersion lens uses oil with 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 (distance between the object on slide and the objective lens, when in focus)

o 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) adjusts the diameter of the cone of light so that it just fills the objective lens

As you close down the diaphragm:1. The light intensity decreases2. Contrast improves3. Depth of field increases4. Limit Resolution (with oil immersion lens)

Resolution (Resolving power) d: Expressed as d d = the smallest distance between two objects which can be seen as separate

d = the diameter of the smallest resolvable objectd = λ / 2 NA

λ = wavelength of lightNA = numerical aperture

To improve resolution, lower d

o d can be decreased by lowering λ or increasing the NA

Morphology of Bacteria: Light Microscopy reveals three principle forms of microorganism:

o More or less spherical organisms:COCCIo Cylindrical organisms: BACILLIo Spiral shaped HELICOIDAL

Incompletely separated cocci may appear in a number of different patterns depending upon the plane in which they divide and how they remain attached:

o Diplococci (pairs) – divide in one planeo Streptococci (chains) – divide in one planeo Tetracocci (tetrads) – divide in two planeso Staphylococci (clusters) – divide in three planes irregularlyo Sarcinae (cuboidal packets) – divide in three planes regularly

Bacilli can appear in a number of different cyclidrical shapeso Coccobacillus are very short and almost appears spherical, but they are just slightly

longer in one direction than the othero Fusiform Bacilli are tapered at the ends, appearing as football like in shapeo Filamentous Bacillary Forms grow in long threds

Staining Procedures:

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: Cationic (basic dyes, positively charged chromophore) – Methylene Blue, Crystal Violet Anionic (acidic dyes, negatively charged chromophore) – Acid fuschin, Congo Red, Nigrosin

Fat Soluble (no charge): Sudan Black stains granules of Poly-B-OH-butyric acid Insoluble Dyes (water insoluble): India Ink (colloid suspension of carbon particles)

Types of Staining Procedure:

1. Negative StainingStains background, not the cell in brightfield microscopy (Not Dark field Microscopy)

Two dyes used:o Nigrosin – a black anionic (negatively) charged dye. The negatively charged dye is

repelled by the negatively charged surface of the bacterial cello India Ink – an insoluble dye (a colloidal suspension of carbon particles) which does not

penetrate the cell surface 2. 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.

3. Differential Staining Staining procedure which causes cells to stain differently based on properties of the cell. Two examples of differential staining:

o Gram stain – A differential stain procedure that causes cells to stain differently based on characteristics of their cell wall. Gram-positive microorganisms have a

higher peptidoglycan and lower lipid content than gram-negative microorganisms. 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. Gram neg. cells are easily decolorized because the ethanol dissolves the high lipid cell wall allowing the crystal violet-iodine complex to readily exit the cell. Gram pos. cells resist decolorization due to the difference in cell wall consistency retaining the crystal violet-iodine complex. The gram neg. cells are then counterstained with saffranin.

Stain Gram + Gram -Primary stain Crystal Violet purple purpleMordant Iodine purple purpleDecolorizer Ethanol purple colorlessCounterstain Saffranin purple pink

o Acid Fast Stain - A differential stain procedure that causes cells to stain differently based on characteristics of their cell wall. Acid Fast microorganisms have a high wax content in their walls, which requires the use of steam to allow dye to penetrate

the cell. Cells are steamed in the presence of carbol 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.

Stain Acid Fast + Acid Fast -Primary stain Carbol Fuschin red red

Steam red redDecolorizer Acid Alcohol red colorlessCounterstain Methylene Blue red blue

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

o 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

o Two important species: tuberculosis and lepraeo Nocardia –

4. Structural Staining Spore Staining

o 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

o Bacillus – Aerobic, gram + rodo Clostridium – Anaerobic gram + rodo Sporsarcinae – Cocci

· Anaerobic green = endospore/free spores Clostridium

· Aerobic green = endospore/free spores of Bacillus· Anaerobic pink = vegetative/sporangia of Clostridium· Aerobic pink = vegetative/sporangia of Bacillus

Stain Spores Non SporePrimary stain Malachite Green Green Green

Steam Green GreenDecolorizer Water Green ColorlessCounterstain Safranin Green Pink

Media and Biochemical Tests:

There are five Methods of Tube Media Preparation:1. Pour = 15 – 20 mL of liquid agar used to pour into a plate2. Broth = 5 – 7 mL of liquid media3. Deep = 5 – 7 mL of media which has solidified in an upright position4. Slant = 5 – 7 mL of media which has solidified at an angled position5. Fermentation Broth = Broth with Durham Tube added

Natural Media – Media composed of complex raw materials whose actual chemical composition is unknown (example: 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) (Selective ONLY)

o Selects for the growth of gram + microorganisms, because Phenylethyl Alcohol is inhibitory to the growth of gram neg. organisms

o Selective/Inhibitory agent: phenylethyl alcohol

o Desoxycholate Agar (DES)(very similar to EMB) (both selective and differential) Selective/inhibitory agent: desoxycholate-bile salt Differential agent: lactose

o Selects for gram neg. microorganism, because Desoxycholate Agar is inhibitory towards the growth of gram + organisms

o 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.

o Lactose (+) red Lactose (-) no color change

Eosin Methylene Blue (EMB) (Selective AND differential)o Selects for gram neg. organisms. o Selective agent/inhibitory agents : eosin and methylene blue (inhibit gram +) o Differentiates lactose +/- microorganisms. Lactose + show a color change (red dye

on the edges of colonies), Lactose – do not show a color change (IMPORTANT NOTE: if the center of the colony is colored but the edge is not so the colony is NOT a lactose fermentor)

o Differential component: lactoseo Bi-products of fermentation: Acid and alcohol (bacteria produce large amounts of

acid)o Can further differentiate Lactose + fermentors based on the amounts of acid produced

during lactose fermentation. If able to ferment lactose then glucose is released (acid produced as bi-product) the acids will attract the dyes into the colony (small amt: red. Large amt: purple/dark)

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

(E.Coli (+) for lactose fermentor) 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 (i.e. bull’s-eye colonies), and will not have a metallic green sheen like Enterobacter.

Blood Agaro Differentiates microorganisms based on their reactions on blood

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

Gamma Hemolysis – No blood hemolysis, no zone of clearing around the colony

Strep is used as the best example of beta hemolysisBiochemical Tests – Tests used to determine physiological characteristics of microorganism, particularly in terms of bacterial enzymes and the chemistry of biooxidation.

Starch Agaro 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.

o (+) (-) Milk Agar

o 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.

o Milk is mostly water* Lipase Plate

o 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 Tubeso 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

Yellow = ACID Yellow + Gas = ACID, GAS Red to dark red = Negative or Alkaline

Methyl Red (MR)

o HCOOH→CO2 + H2

o Tests for a Mixed Acid Fermentoro Mixed Acid Fermentors produce drastic amounts of acid from the fermentation of

sugars. This acid ultimately results in the lowing of the pH below 5.1, so when the indicator methyl red is added to the culture the methyl red remains red.

o Escherichia = MR +, if it dissipates or turns orange then it is NOT a mixed acid fermentor

o

Voges-Proskauer (VP) (Sugar enriched media) o HCOOH → acetyl methyl carbinol →2,3 butanediolo Tests for 2,3 butanediol fermentoro 2,3 butanediol fermentors produce less acid and more neutral products than Mixed

Acid Fermentors. Because acetyl methyl carbinol (acetoin) is easier to detect than 2,3 butanediol, acetoin is tested for when determining if a microorganism is a 2,3 butanediol producer. Barrit’s reagents, also known as VPI (alpha-naphthol) and VPII (KOH) is added to the test culture. When oxygen is present KOH will react with Acetoin to produce a brick red color and indicate that the microorganism is a 2,3 butanediol producer. Alpha-naphthol is used to intensify the red color.

o Enterobacter = VP +

o Catalase

o 2H2O2 2H2O and O2

o Hydrogen Peroxide is produced during oxygen utilization and must therefore be eliminated since hydrogen peroxide is toxic. Catalase is an enzyme which converts hydrogen peroxide to water and oxygen, and can be tested for by merely adding H2O2 to the culture of question and look for the production of oxygen bubbles being produced.

o

Oxidase: Oxidase is an enzyme which can oxidize aromatic amines to form colored productso Aromatic amine used to test for oxidase is dimethyl-p-phenylenediamine

hydrochloride which when in the presence of oxidase will turn a dark blue black color.

Nitrate o NO3

- + 2e +2H+ NO2 + H2Oo NO2 → →→ N2 + NH3 other productso Tests for the ability of microorganisms to reduce Nitrate (nitrate reductase)o Organisms are grown in Nitrate broth which contains nitrates (NO3

-), o Reagents Nitrate I (Sulfanilic Acid) and Nitrate II (dimethyl-alpha-

naphthylamine) are added to the broth,o if NO2

-, a product of nitrate reduction, is present the broth will appear red. If the broth is red at this point the microorganism is said to be Nitrate pos.

o If the broth is not red, Zinc is added to the tube. Zinc is a catalyst which will convert NO3

- to NO2-. Since the nitrate reagents are still in the tube, if NO3

- was still present the broth will turn red.

o If the broth turns red after addition of zinc, the microorganisms are said to be Nitrate neg. If the broth does not turn red, the NO3

- of the Nitrate broth was reduced by the microorganism to products other than NO2

-, and is therefore said to be Nitrate pos.

Tryptophan (Indole) o Tryptophan Pyruvic Acid and Indole o Tests for the enzyme tryptophanase which converts trytophane to indole and

pyruvic acid. Indole can easily be tested for by adding Kovac’s Reagent (p-dimethylaminobenzaldehyde, amyl or butyl alcohol, and HCl) which will appear red in the presence of the product indole.

Ureao Urea 2NH3 + CO2

o Tests for the enzyme Urease which converts urea to ammonia and CO2. Urea broth contains the substrate urea and the pH indicator phenol red. When ammonia is released the pH of the solution increases and once the pH is above 8.1 the phenol red indicator will appear Red. The red color indicates a positive test for Urease.

o Proteus = Urease +

o Hydrogen Sulfide Production (H2S)

o Cysteine H2S + Amino Acrylic Acid Imino Acid Pyruvic Acid + NH3

o Tests for the enzyme cysteine desulfurase which removes the sulfur side chain from cysteine to produce H2S, which when in the presence of iron salts (contained in Klinger’s Iron Agar and SIM medium) forms a black precipitate. This Black Precipitate is a positive test for cysteine desulfurase (aka H2S positive)

o Proteus = H2S positive

Simmons Citrateo Tests for the ability of a microorganism to utilize citrate as the sole carbon

source.

o If a microorganism can use citrate as the sole carbon source the microorganism will grow on the bacterial medium and the media will turn a deep Prussian Blue color. Growth on the media and the appearance of the Prussian Blue color are both indications of a citrate positive microorganism.

o 2 positive results o –Growth AN/OR blue color, growth alone can be a positive test (most obvious

response)

o

SIMo Tests for Sulfur (H2S production), Indole, and Motilityo H2S positive = black precipitate when it reacts with IRONo Indole positive = Kovacs Reagent turns red after addition (tilt the tube and

make sure the LIQUID is positive since pseudomonas gives a false positive)

o Motility positive = growth away from inoculation line (appears as cloudiness in tube If black is also throughout the tube then we can assume the organism is also MOTILE)

Phenylalanine (PPA) (one of the 20 amino acids)o Phenylalanine Phenylpyruvic Acid (PPA) + NH3

o Tests for the presence of the enzyme phenylalanase/phenylalanine deaminase which converts Phenylalanine to PPA and NH3. To test for the presence of PPA ferric chloride is added to the media. Ferric Chloride in the presence of PPA will appear a deep green color on the surface of the slant. This green color is indicative of a positive test for phenylalanase.

(+) (-)

Litmus Milko Tests for Lactose fermentation, reduction of litmus, presence of caseinase, and the

deamination of amino acids to produce NH3

o Litmus Milk contains the pH indicator Litmus (neutral is purple , acid is pink and alkaline/basic is blue ) and powdered milk. From this mixture multiple different Litmus Milk results can be obtained:

Acid Reaction – Pink Liquid due to drop in pH from the fermentation of lactose

Acid Curd Reaction – Pink Solid due to acid production and coagulation of proteins causing the solid formation. Bottom may be white because

Reduction – Litmus is reduced and is caused to be colorless and the tube appears white since only the Milk remains.

Alkaline Reaction – Blue liquid which is usually caused when protein breakdown produces amino acids that are deaminated and release ammonia.

Peptonization/Proteolysis – Clearing of medium (may be brown or amber) caused by enzyme caseinase which breaks down the white protein casein in milk.

Multiple reactions can also be observed: ex Acid Curd Reduction – Looks like Acid Curd but the tube turns white except for a small region at the top where oxygen reoxidizes the litmus to the colored form, which is red.

Kliger’s Iron Agar (KIA)Tests for ability to ferment glucose and or lactose, tests for H2S production, and can also be used to test for gas production.

Glucose and Lactose fermentation is determined using a pH indicator which begins red and will turn yellow in the butt of the tube if glucose is fermented, and will cause the slant to turn yellow if lactose is able to be fermented. If a bacteria contains the enzyme cysteine desulfurase, a black precipitate

will form Gas production can be determined by cracks and or the lifting of the slant

off the bottom of the tube

corresponding tube no. above 1 2 3 4* 5deamination of amino acids (aerobic alkaline rx.) + + + + +

glucose fermentation (minor acid rx.) – + + + +

lactose fermentation (major acid rx.) – – – + +

H2S production (black color) – – + – +**

*Note that KIA is ideally read ~18 hrs after inoculation and the lactose reaction should be read from the bottom of the slant as the tip of the slant may revert back to red as the inoculation ages beyond 18-24 hours.

OF Glucose (oxidative fermentative) o Set of tests used to determine if a bacteria can use glucose in an oxidative

(aerobic) or fermentative (anaerobic) condition.o Two tubes are inoculated, with one of the tubes covered in mineral oil to prevent air

from reaching the media. Media contains pH indicator (Brom Thymol Blue) which turns yellow if the glucose is utilized and acids are produced.

o Results: Open Tube: Yellow, Closed Tube: Uninoculated color = Incompletely Oxidative (O) Open Tube: Yellow, Closed Tube: Yellow = Strictly Fermentation (F) Open Tube: Uninoculated color, Closed Tube: Uninoculated color = Strictly

Oxidative Open Tube: Uninoculated color, Closed Tube: Yellow = Facultative

Glucose +Lactose +Desulferase +

Glucose +Lactose -Desulferase +

Glucose +Lactose +Desulferase –

Glucose +Lactose –Desulferase –

Glucose –Lactose –Desulferase –

IMViCo Set of four tests that are used to differentiate between Escherichia coli and

Enterobacter aerogeneso Indole, Methyl Red, Voges-Proskauer, and Citrate

I M V CE. coli + + - -E. aerogenes - - + +

Other Tests and Media: Motility Media

o Tests if the bacteria are motile or not

Incompletely Oxidative

Strictly Oxidative

Facultative Strictly Fermentative

o Contains Tetrazolium chloride, a growth indicator which turns red in the presence of growing bacteria. Therefore Red color away from the inoculation line is an indicator of growth. Prodius merabillis(+)Staphylococcus aureus(-)

o Red color is only a growth indicator and indicates that tetrazolium chloride is reduced, the red color does not mean motility. Where the red color appears is an indication of motility, not the appearance of red.

o If tetrazolium chloride is not present, cloudiness throughout the tube (+) for motility. (SIM tube is capable to determine this)

Bismuth Sulfide Agar (BSA) o A dull green color; Salmonella typhi produces a black or very dark brown color

Brilliant Green Agar (BGA)o Differential for lactose/sucrose fermentation

Lactose/ sucrose fermenting organisms produce yellow/ green or yellow colonies and turn the surrounding media yellow/green

Non - lactose/ sucrose fermenting organisms produce opaque red/ pink/ white colonies and turn the surrounding media red

SS Agar

o On this medium Salmonella usually produces a black colony, Shigella a colorless colony & all lactose positive colonies appear red