Lab Practical

Simple Microscope

One lens

Compound 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)


Arm, Base structural parts of the microscope which support the
basic frame.


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

Lens System

Oculars, Objectives, and Condenser


eyepiece lenses (usually 10x magnification)


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 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 (distance between the object on slide and the objective lens, when in focus) decreases


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

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 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 = ? / 2 NA
? = wavelength of light
NA = numerical aperture

To improve resolution,

lower d
d can be decreased by lowering ? or increasing the NA

Light Microscopy reveals three principle forms of microorganism:

More or less spherical organisms: COCCI
Cylindrical organisms: BACILLI
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:

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

Bacilli can appear in a number of different cyclidrical shapes

Coccobacillus are very short and almost appears spherical, but they are just slightly longer in one direction then the other
Fusiform Bacilli are tapered at the ends, appearing as football like in shape
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 affin

Cationic Dye

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

Anionic Dye

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

Fat Soluble Dye

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

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)
Two dyes used: Nigrosin and India Ink


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

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 th

Differential Staining

Staining procedure which causes cells to stain differently based on properties of the cell.
Two examples of differential staining: Gram Stain and Acid Fast Stain

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 c

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 a

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

Mycobacterium and Nocardia


Do not gram stain well if mature because of high wax content within walls, if young appear as gram + tapered rods that sometimes fragment
Two important species: tuberculosis and leprae

Structural Staining

Spore Staining
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

Spore Staining

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, Clostridium, and Sporsarcinae.


Aerobic, gram + rod


Anaerobic gram + rod



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

There are five Methods of Tube Media Preparation:

Pour, Broth, Deep, Slant, and Fermentation Broth


15 - 20 mL of liquid agar used to pour into a plate


5 - 7 mL of liquid media


5 - 7 mL of media which has solidified in an upright position


5 - 7 mL of media which has solidified at an angled position

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. 2 Types: Selective Media and Differential Media

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.

Phenylethyl Alcohol Agar (PEA)

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

Desoxycholate Agar (DES)

Selects for gram neg. microorganism, because Desoxycholate Agar is inhibitor towards the growth of gram + organisms
Differentiates for lactose fermentors (lactose + microorganisms from lactose negative). Lactose fermentors produce acid which precipitates

Eosin 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 fermentatio

Blood Agar

Differentiates microorganisms based on their reactions on blood.
Different reactions on blood: Gamma Hemolysis, Beta Hemolysis

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

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 aro

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 teste

3 Types of Sugar Fermentation Tubes

Yellow, Yellow + Gas, and Red to dark red



Yellow + Gas=


Red to dark red=

Negative or Alkaline

Methyl Red (MR)

Tests for a Mixed Acid Fermentor
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

Voges-Proskauer (VP)

HCOOH ? acetyl methyl carbinol ?2,3 butanediol
Tests for 2,3 butanediol fermentor
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 butan


2H2O2 ? 2H2O and O2
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 addi


Oxidase is an enzyme which can oxidize aromatic amines to form colored products
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.


NO3- + 2e +2H+ ? NO2 + H2O
NO2 ? ?? N2 + NH3 other products
Tests for the ability of microorganisms to reduce Nitrate
Organisms are grown in Nitrate broth which contains nitrates (NO3-), Reagents Nitrate I (Sulfanilic Acid) and Nitrate II (dimethyl-alpha-

Tryptophan (Indole)

Tryptophan ? Pyruvic Acid and Indole
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 wil


Urea ? 2NH3 + CO2
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

Hydrogen Sulfide Production (H2S)

Cysteine? H2S + Amino Acrylic Acid?Imino Acid? Pyruvic Acid + NH3
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 SI


Tests for Sulfur (H2S production), Indole, and Motility
H2s positive = black precipitate
Indole positive = Kovacs Reagent turns red after addition
Motility positive = growth away from inoculation line (appears as cloudiness in tube)

Simmons Citrate

Tests for the ability of a microorganism to utilize citrate as the sole carbon source.
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. Gro

Phenylalanine (PPA)

Phenylalanine? Phenylpyruvic Acid (PPA) + NH3
Tests for the presence of the enzyme phenylalanase 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 wil

Litmus Milk

Tests for Lactose fermentation, reduction of litmus, presence of caseinase, and the deamination of amino acids to produce NH3
Litmus Milk contains the pH indicator Litmus and powdered milk. From this mixture multiple different Litmus Milk results can be o

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


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.


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 re


Set of four tests that are used to differentiate between Escherichia coli and Enterobacter aerogenes
Indol, Methyl Red, Voges-Proskauer, and Citrate

Kliger's Iron Agar (KIA)

Tests for ability to ferment glucose and or lasctose, 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 t

If a bacteria contains the enzyme cystein 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

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 in some species.

Glucose -
Lactose -

Pseudomonas Alkalingenes

Glucose +
Lactose -

Salmonella Shigella Proteus

Glucose +
Lactose +


OF Glucose

Set of tests used to determine if a bacteria can use glucose in an oxidative (aerobic) or fermentative (anerobic) condition.
Two tubes are inoculated, with one of the tubes covered in mineral oil to prevent air from reaching the media. Media contains pH i

Incompletely Oxidative (O)=

Open Tube: Yellow, Closed Tube: Uninoculated color

Strictly Fermentation (F)=

Open Tube: Yellow, Closed Tube: Yellow

Strictly Oxidative=

Open Tube: Uninoculated color, Closed Tube: Uninoculated color


Open Tube: Uninoculated color, Closed Tube: Yellow

Motility Media

Tests if the bacteria are motile or not
Contains Tetrazolium chloride, a growth indicator which turn red in the presence of growing bacteria. Therefore Red color away from the inoculation line is an indicator of growth.
Red color is only a growth indicato

Bismuth Sulfide Agar (BSA)

A dull green color; Salmonella typhi produces a black or very dark brown color

Brilliant Green Agar (BGA)

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

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

Desoxycholate Citrate

Selects for gram -, lactose - microorganisms
Some Lactose + colonies do grow but they will appear Red


Incubate bacteria in small tube of plasma overnight
If plasma becomes clumpy and or solidifies, then bacteria are coagulase positive
Test is only valid on gram + staphylococcus like bacteria since gram negative bacteria are able to provide false positive

Phenol Red Mannitol Salt Agar (MSA)

Selects for Staphylococcus due to high salt concentration 7.5%
Medium is red, but plate and colonies will turn yellow if organisms are mannitol positive

Staphylococcus 110 Medium

Contains Mannitol and 7.5% NaCl, but lacks Phenol Red as in MSA plate
Selects for Staphylococcus and allows for development of natural colony pigment formation unlike in MSA


Tests for exoenzyme DNase which is able to hydrolyze DNA
Zones of clear around streaks either before or after addition of 0.1N HCl is a positive result for the presence of DNase

M-staphylococcus broth

Enriched media containing 10% NaCl, which selects for Staphylococcus since Staphylococcus prefer the higher salt concentration, which inhibits most other organisms

Endo Agar

Selects for gram -
Diferential for lactose, lactose + = red colonies and surrounding medium
Coliforms produce a golden metallic golden sheen

Gram Positive Pyogenic Cocci:

Medical Microbiology is primarily concerned with the isolation and Identification of pathogenic organisms. One of the most frequently encountered sections of pathogenic bacteria are the gram positive cocci. Of these bacteria the two Genus's we will focus


Found in nasal membranes, the hair follicles, the skin, and perineum
Most strains are penicillin resistant, which can cause epidemiology problems since 90% of healthcare workers carry Staphylococcus
Divide in multiple planes and there appears as irregular


Found in pharynx, on surfaces of the teeth, saliva, shin, colon, rectum, and vagina
Divide in only one plain and therefore appear as chains of cocci
Streptococci of greatest medical significance are: S. pyogenes, S.pneumoniae, and S. agalactiae

Isolation and Identification of Staphylococcus and Streptococcus

Consists of a 4 day process

Day 1:

Swab Nose, Throat, and a Fomite
Streak swab onto Blood Agar Plate as shown
Place swabs into m-Staphylococcus broth

Day 2:

Use the three m-Staphylococcus broths to inoculate two Staphylococcus medium 110 (SM110) and two Mannitol Salt Agar (MSA) Plates
Select a Beta hemolytic staphylococcus from the Blood Agar plate and inoculate third SM 110 and MSA plate

Day 3:

Using the SM110 and MSA plates from last period to identify 3 presumptive Staphylococcus colonies by their growth on the SM110 and MSA plates as well as the cluster formation of the gram + cocci.
Inoculate each of these 3 colonies onto/int

Day 3:

Using the Blood Agar plates from last period identify 3 Alpha or Beta hemolytic Streptococcus by identifying the chain formation of the gram + cocci.
Inoculate each of these 3 colonies into a Nitrate broth and perform a catalase test on eac

Day 4:

Examine the results of the Nitrate Broth, DNase, and Coagulase inoculations made last period and identify the species of Staphylococcus.
Examine the results of the Nitrate Broth inoculations made last period and identify the

Gram Negative Intestinal Pathogens:

Gram-negative intestinal pathogens are a major concern for public health since the two main pathogens Salmonella and Shigella have the ability to cause enteric fevers, food poisoning, dysentery, and even typhoid fever. Salmonella has over 2200 serotypes i

Gram Negative Intestinal Pathogens:

Public Health Laboratories routinely test for the presence of the Gram (-) pathogens by the isolation and identification of Salmonella and Shigella from feces. This makes isolation and identification difficult due to the presence of Escherichia, Proteus,

Gram Negative Intestinal Pathogens Procedure

Consists of a 3 day process

Day 1:

Perform Isolation streaks of the Salmonella containing mixture onto each of the selective/differential media provided.

Day 2:

From the five selective/differential plates select 7 isolated colonies that are presumptive for being Salmonella based on their appearance and inoculate each into a SIM, Urea, and KIA media.

Colonies to be selected:

EMB: Lactose (-) bacteria (colonies do not change color)
DES citrate: Lactose (-) bacteria (colonies do not change color)
BGA: Lactose (-) (colonies appear pink/white surrounded by red media)
SS agar: Black colonies
BSA: Black colonies

Day 3:

Determine the identity of each of the selected colonies.

Standard Plate Count:

To determine a bacterial population count (the number of organisms that are present in a given unit of volume) several methods are available, the one of the simplest method being the standard plate count. That standard plate count is accomplished by dilut

Standard Plate Count:

Advantages of the Standard Plate Count over other Bacterial Plate Count Methods is the fact that it has a very basic principle and technique that requires very minimal amount of equipment but still provides excellent results. The Disadvantages of the Stan

Rules for Standard Plate Count:

1.Pick the plate that contains between 30 - 300 bacterial colonies to count
2.Calculate all the dilution factors (DF) between the counted plate and the original culture
a.DF = amount added / (amount added + amount already there) DF = A/(A+B)
b.DF for plat

Example of Standard Plate Count:

1.Select plate with 32 colonies
2.Plated dilution = 1ml/1ml = 1 all other dilutions = 1/(1+99) = 1/100
3.Bacterial plate count = 32 x 1 x 100 x 100 x 100 = 3.2 x 107 bac/ml

Direct Microscopic Count:

Direct Microscopic Counts are usually performed in milk to determine the quality in a much shorter time than a standard plate count. The microscopic count is accomplished by staining a measured amount of milk that has been spread over a known area (usuall

Direct Microscopic Count:

High quality milk will have very few microorganisms per field, necessitating the examination of many fields. A slide of poor quality milk will reveal large numbers of bacteria per field, requiring the examination of fewer fields. An experienced technician

Direct Microscopic Count:

In addition to being much faster than a Standard Plate Count, the direct microscopic count has two other distinct advantages. First of all, it will reveal the presence of bacteria that does not form colonies on an agar plate at 35�C (thermophiles, psychro

Procedures for Milk Staining:

1.Shake the milk to disperse organisms and break up large clumps of bacteria
2.Transfer 0.01ml of milk to one square of the breed slide
3.Spread the milk to the edges of the indentions in the breed slide
4.Allow the slide to air-dry
5.Steam the slide over

Calculation of the number of microorganisms in a suspension:

Number organisms per ml = (Ave. # bacteria/field of view) X (Microscopic Factor) X 1/(Dilution Factor)

Ave. # bacteria /field =

the average bacteria counted per view of the microscope

Dilution factor =

amount of milk added to breed slide/1 ml

Standard amount of milk added to bread slide =

0.01ml; therefore standards dilution factor = 0.01ml/1ml = 1/100

Microscopic Factor =

Area of Film/Area of Microscopic Field

Area of the Film =

area of indention in breed slide = 1cm2 = 100mm2

Area of the Microscopic Field=


To determine the radius of the microscopic field,

a stage micrometer is used. The stage micrometer is placed under the microscope and the number of divisions from one edge of the field of view to the other is counted, determining the diameter of the field of view.

1 division using the a stage micrometer =

0.01 mm

Radius =

Diameter / 2

An average of 45 bacteria per field were counted, when 0.01 ml was added to a 1cm2 indention on a breed slide, the diameter of the field of view was determined to be 0.20mm.

Organisms/ml = (Ave. # bacteria/field) x (Microscopic Factor) x (Dilution Factor)
Organisms/ml = (45 bac/field) x (100mm2 / ? (0.10mm)2) x 1/(0.01ml / 1ml)
Organisms/ml = (45) x (100mm2 / (3.14 x 0.01mm2) x (100)
Organisms/ml = (45) x (3185) x (100)

Bacterial Examination of Water:

Water is routinely examined for fecal contamination, to ensure sanitary water suplies. Fecal contamination is identified by finding coliforms in the water. A coliform is defined as a facultative anaerobe that ferments lactose to produce gas, and is a gram

Presumptive Test:

A series of 9-12 tubes of lactose broth used to identify if there are any bacteria in the water that are lactose fermenting gas producing. These tests are also used to determine the most probable number (MPN) of coliforms present per 100 ml of water.

Confirmed Test:

EMB or Endo Agar plates are inoculated from gas positive lactose broths. On EMB agar, coliforms produce small colonies with dark centers. On Endo agar, coliforms produce reddish colonies. These results indicate that the identified colonies are grem negati

Completed Test:

Lactose positive gram negative colonies are selected and inoculated into lactose broth and onto nutrient agar slants. If gas is produced in the lactose broth and staining reveals that the bacteria are gram negative rods and non-spore-forming then the wate


To confirm that the positive coliform test is due to E.coli and not E.aerogenes, the IMViC set of tests must be conducted.

Memebrane Filter Method:

Method of examining water for bacterial contamination by passing the water through a filter. The filter captures all the bacteria (does not retain viruses) in the water but allows the water to pass. The filter is then placed into a differential medium to

mENDO Agar (pink) shows...

total coliforms by a golden sheen

mFc Agar (light blue) shows...

fecal coliforms as darker blue colonies on the agar

Membrane Filter method has advantages over the multiple tube method:

Higher degree of reproducibility
Greater sensitivity
Shorter time requirement

Antiseptic Evaluation:

Consists of 3 methods: Filter Paper Disk Method, Kirby-Bauer Method, and Minimum Inhibitory Concentration (MIC)

Filter Paper Disk Method

Used to compare antiseptics based on their bacteriostatic properties
Relative effectiveness is measured by the size of the zone of inhibition (measured from edge of plate to edge of inhibition zone) and can be compared quantitatively against other substan

Kirby-Bauer Method

Used to compare effectiveness of antiseptics, both antibiotics (made naturally) and drugs (man made) which is done under a standardized system.
Methodiology standardizes: diffusibility of the agent, size of the inoculum, type of medium, and many other fac

Minimum Inhibitory Concentration (MIC)

Establish the minimum concentration of an antiseptic needed to inhibit the growth of a test microorganism

MIC Procedures:

Make dilutions of the antiseptic needed for testing
Tubes 6-9 are controls
Tubes 1-5 are the antimicrobial test tubes
The MIC is the most dilute (the minimal concentration) tube which prevents growth of the test organism