Académique Documents
Professionnel Documents
Culture Documents
Students are responsible for all the material in this lab packet.
Last reviewed: January 2016
1. Backpacks, purses, and other personal effects will be placed on the rack near
lab entrance when you first come in to lab. Paper and writing utensils are
2.
3.
4.
5.
6.
7.
Date: _____________________
Lab section: ________________
MICROSCOPE BASICS
BACTERIAL MORPHOLOGY
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 plane
o Streptococci (chains) divide in one plane
o Tetracocci (tetrads) divide in two planes
o Staphylococci (clusters) divide in three planes irregularly
o Sarcinae (cuboidal packets) divide in three planes regularly
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 fuchsin, Congo red, Nigrosin
Fat Soluble (no charge): Sudan black stains granules of Poly--OH-butyric acid
Insoluble Dyes (water insoluble): India ink (colloid suspension of carbon particles)
Types of Staining Procedure:
1. Negative Staining
Stains the background and not the cell in bright-field microscopy (not the same thing as dark field
microscopy).
Does not require heat fixation, therefore does not distort size or shape of cells.
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 cell.
o 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.
Two commonly used 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
Acid Fast Stain 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 fuchsin 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.
Primary stain
Decolorizer
Counterstain
o
Stain
Acid Fast (+) Acid Fast (-)
Carbol fuchsin
red
red
Steam
red
red
Acid alcohol
red
colorless
Methylene blue
red
blue
Two genera of acid fast organisms (all other genera are non-acid fast):
i. Mycobacterium they do not Gram-stain well if mature because of high wax content
within walls. If the colony is young it will appear as Gram-positive tapered rods that
sometimes fragment.
Gram Stain 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. Gramnegative cells are easily decolorized because the ethanol dissolves the high lipid cell wall allowing the
crystal violet-iodine complex to readily exit the cell. These cells are then counterstained with Safranin.
Gram-positive cells resist decolorization due to the difference in cell wall consistency retaining the
crystal violet-iodine complex.
Primary stain
Mordant
Decolorizer
Counterstain
4.
Stain
Crystal violet
Iodine
Ethanol
Safranin
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 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.
o Endospores appear as a green center within a pink sporangium
o Free spores appear as small green oval bodies
o Three genera of spore forming organisms
Bacillus aerobic, Gram-positive rod
Aerobic green = endospore/free spores of Bacillus
Aerobic pink = vegetative/sporangia of Bacillus
Clostridium anaerobic, Gram-positive rod
Anaerobic green = endospore/free spores Clostridium
Anaerobic pink = vegetative/sporangia of Clostridium
Sporosarcina cocci
Primary stain
Decolorizer
Counterstain
Stain
Malachite green
Steam
Water
Safranin
Spores
green
green
green
green
Non Spore
green
green
colorless
pink
3.
4.
5.
Lactose fermenters produce acid, which precipitates the bile salts in the media and absorbs the
neutral red dye, therefore appearing red/pink.
Non-fermenters do not do this and do not appear red/pink but will form colorless colonies.
Blood Agar
o Differentiates microorganisms based on their reactions on blood. Shows presence or absence of an
exoenzymes known as hemolysins that can break down hemoglobin in blood.
Gamma () hemolysis: no blood hemolysis, no zone of clearing around the colony.
Beta () hemolysis: complete blood hemolysis and complete clearing around the colony.
Hemoglobin is completely degraded.
Alpha () hemolysis: partial blood hemolysis and partial clearing around colony. Partial
clearing sometimes appears green due to partial reduction of hemoglobin in blood. Greenish
color is due to a partial breakdown product of hemoglobin called biliverdin.
Biochemical Tests:
Tests used to determine physiological characteristics of microorganism, particularly in terms of bacterial enzymes
and the chemistry of bio-oxidation. Biochemical tests on agar plates often (but not always) look for the presence of
exoenzymes which are enzymes secreted by the bacteria into their environment to break down macromolecules,
such as proteins and carbohydrates, into monomeric subunits small enough to be transported into the bacterial cell.
Endoenzymes are enzymes active inside the bacterial cell to metabolize substrates inside the bacterial cell, and can
be detected in both solid media and broths.
1.
2.
3.
4.
Starch Agar
Tests for the presence of the exoenzyme amylase, which hydrolyzes starch to simple sugars.
o
These simple sugars can then be transported inside the cell.
Iodine
is
added to the starch plate and appears blue/black when iodine forms a complex with starch.
o
If amylase is present, starch will be hydrolyzed and the blue/black to purple color will not be observed
o
around the colonies.
Milk Agar
Tests for the presence of the exoenzyme caseinase, which hydrolyzes casein (a predominant protein in
o
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
o
and become clear around the caseinase positive colonies.
Lipase Plate
Tests for the presence of the exoenzyme lipase, which hydrolyzes fat to form glycerol and fatty acids.
o
The production of the fatty acids lowers the pH just enough to produce a dark blue precipitate when a
o
microorganism is lipase positive.
Spirit blue dye is the indicator in this test.
o
Positive test shows an intensification of blue color around the colony.
o
Sugar Fermentation Tubes
Used to determine if a microorganism can ferment a particular sugar.
o
The fermentation tubes contain the sugar of interest (glucose, lactose, or mannitol), a pH indicator
o
(phenol red) and a Durham tube.
If a microorganism is able to ferment the sugar being tested the result of the fermentation will lead to
o
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
o
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
o
the testing sugar, releasing ammonia into the medium. An alkaline reaction is indicated by the
darkening of the red pH indicator color into a hot pink/cerise color.
Yellow = acid
Red = negative
Cerise = alkaline
Methyl Red (MR)
HCOOH CO2 + H2
o
Tests for a mixed acid fermenter which produce drastic amounts of acid from the fermentation of
o
sugars.
This acid ultimately results in the lowing of the pH below 5.1, so when the indicator methyl red is
o
added to the culture the methyl red remains red.
Red is the only true indicator of a positive result.
Escherichia is MR positive.
o
5.
6.
7.
8.
9.
Voges-Proskauer (VP)
HCOOH acetyl methyl carbinol (AMC)/acetoin 2,3-butanediol
o
Tests for 2,3-butanediol fermenters which produce less acid and more neutral products than mixed acid
o
fermenters.
Because AMC is easier to detect than 2,3-butanediol, AMC is tested for when determining if a
o
microorganism is a 2,3-butanediol producer.
Barritts reagents, also known as VPI (-naphthol) and VPII (KOH), are added to the test culture.
o
When oxygen is present, KOH (VP II) will react with AMC oxidizing it to diacetyl, which reacts with
o
a guanidine-containing compound in the media to produce a brick red color, indicating that the
microorganism is a 2,3-butanediol producer.
-naphthol (VP I) is used to intensify the red color by catalyzing the oxidation of AMC by
KOH (VPII).
Enterobacter is VP positive.
o
Catalase
Tests for the presence of catalase, which converts hydrogen peroxide to water and oxygen.
o
2 H2O2 2 H2O + O2
Hydrogen peroxide is produced during oxygen utilization and must therefore be eliminated since
o
hydrogen peroxide is toxic to the cell.
Enzyme presence can be tested for by adding 3% H 2O2 to the culture and looking for the production of
o
oxygen bubbles.
Bubble production is a positive result.
Oxidase
Cytochrome c oxidase is an enzyme which can oxidize aromatic amines to form a colored product.
o
The aromatic amine used to test for oxidase is dimethyl-p-phenylenediamine hydrochloride which in
o
the presence of oxidase will turn a dark blue-black color.
Nitrate
Tests for the enzyme nitrate reductase (nitrase), which can reduce nitrate (NO3-) to nitrite (NO2-) in a
o
single step.
NO3- + 2e- + 2H+ NO2- + H2O
Nitrate I (sulfanilic acid) and Nitrate II (dimethyl--naphthylamine) react with NO 2 to produce a brick
o
red color.
This is a positive result.
If nitrate is not used and is residual, then zinc powder can catalyze the reaction instead.
o
A red result after Zn addition will show that nitrate was not used because the organism lacks
nitrase.
No color after addition of reagents, but a red color after Zn, is a negative result.
Some microorganisms have nitrate reductase as well as nitrite reductase and other reductases.
o
Nitrate will be reduced to either ammonia (NH 3) or nitrogen gas (N2) in a multistep process
known as denitrification.
NO2 N2 + NH3 and other products
Reagents I and II followed by Zn addition will not form a red color since both nitrate and nitrite have
o
been consumed.
Indole can easily be tested for by adding Kovacs Reagent (p-dimethylaminobenzaldehyde (DMABA)
o
and HCl dissolved in amyl or butyl alcohol).
A positive result is when the Kovacs reagent interacts with indole.
o
11. Urea
Tests for the enzyme urease, which converts urea to ammonia and carbon dioxide.
o
Urea 2 NH3 + CO2
Proteus
is
urease positive.
o
12. Hydrogen Sulfide Production (H2S)
It tests for the enzyme cysteine desulfurase which removes the sulfur side chain from cysteine to
o
produce H2S, which when in the presence of iron salts (contained in Klingers Iron Agar and SIM
medium) forms a black precipitate.
Cystein H2S + amino acrylic acid imino acid pyruvic acid + NH3
Proteus
is
H2S positive.
o
13. SIM
Tests for Sulfur reduction (H2S production), presence of Indole, and Motility.
o
H2S positive = black precipitate
o
Black precipitate is formed when the produced H2S combines with iron from ferrous
Motility positive = growth away from inoculation line (appears as cloudiness in tube)
o
14. Simmons Citrate
Tests for the ability of a microorganism to utilize citrate as the sole carbon source.
o
Media contains ammonia salts (sole nitrogen source), citrate (sole carbon source), and bromothymol
o
blue (indicator).
If a microorganism can use citrate as the sole carbon source the microorganism will grow on the
o
bacterial medium and the media will turn a deep Prussian blue color.
Growth and the appearance of the Prussian blue color are indications of a citrate positive
microorganism.
Metabolism of citrate production results in production of bicarbonate and carbonate, raising
the pH.
15. Phenylalanine (PPA)
Tests for the presence of the enzyme phenylalanase, which removes the amine group (NH 2) from
o
phenylalanine, producing phenylpyruvic acid (PPA) and NH3.
Phenylalanine PPA + NH3
Ferric chloride (FeCl3) is added to the media to test for the presence of PPA.
o
In the presence of PPA, ferric chloride will appear a deep green color.
o
A deep green color is indicative of a positive test for phenylalanase.
b) Acid curd reaction: pink solid due to acid production and coagulation of proteins causing the
formation of a solid.
c) Reduction: litmus is reduced and it becomes colorless; the tube appears white since only the
milk remains. Color may remain at top of media where oxygen can oxidize the litmus and
restore its color.
d) Alkaline reaction: appears as a blue liquid that is usually caused when protein breakdown
produces amino acids that are deaminated and release ammonia, increasing the pH in the tube.
e) Peptonization/Proteolysis: clearing of the medium (may be brown or amber) caused by
enzyme caseinase, which breaks down the white protein casein in milk.
More than once reaction can be observed in a single tube:
Example: 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 (red) form.
17. Gelatin
o
o
o
Tests for the presence of the gelatinase enzyme, which hydrolyzes gelatin to amino acids.
Gelatin is a protein that solidifies at lower temperatures.
This tube is stab inoculated and then placed on ice for several minutes after incubation. After
incubation on ice the media may be:
Liquid, this is a gelatinase-positive result (gelatin is hydrolyzed)
*** For the lab midterm you are responsible for knowing everything before this point. PowerPoints, lab lectures,
and whiteboards are all fair game for the test. ***
Other Tests/Media:
18. IMViC
Set of four tests that are used to differentiate between Escherichia coli and Enterobacter aerogenes
o
Indol, Methyl Red, Voges-Proskauer, and Citrate
o
E. coli
E. aerogenes
I
+
-
M
+
-
Vi
+
C
+
RDS
is
read
slant over butt, meaning that the results from the slant are read first and then the results
o
are read from the butt of the tube.
Example: red slant with yellow butt = glucose-positive, lactose-negative
RDS should ideally be read within 24-48 hours after inoculation. After 48 hours the organism could
o
begin to break down the peptone present in the media, which will raise the pH and cause the slant to
revert back to red. This would be a false negative for lactose fermentation.
10
11
KIA differs from RDS due to the addition of iron salts to the media (see SIM above).
Glucose and/or lactose fermentation is determined using a pH indicator (phenol red) which begins red
o
and will turn yellow in the butt of the tube if glucose is fermented. It will cause the slant to turn yellow
if lactose is able to be fermented.
If the bacteria contains the enzyme cysteine desulfurase, a black precipitant will form from
the tube.
KIA is ideally read ~18 hours after inoculation and the lactose reaction should be read from the bottom
o
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.
12
If plasma becomes clumpy and or solidifies, then bacteria are coagulase positive
Test is only valid on Gram-positive staphylococcus-like bacteria since Gram-negative bacteria are able
to provide false positive reactions from a non-coagulase like mechanism.
Phenol Red Mannitol Salt Agar (MSA)
Selects for Staphylococcus due to high salt concentration 7.5%
o
Medium is red, but plate and colonies will turn yellow if organisms are mannitol-positive.
o
Staphylococcus 110 Medium
Contains mannitol and 7.5% NaCl, but lacks phenol red as in MSA plate.
o
Selects for Staphylococcus and allows for development of natural colony pigment formation unlike in
o
MSA.
DNase
Tests for the exoenzyme DNase, which is able to hydrolyze DNA into free nucleotides.
o
Methyl green dye in media complexes with intact DNA in the media. Breakdown of DNA displaces
o
this dye, and forms a clear area.
Zone of clearance around the streak is a positive result for the presence of DNase.
o
m-Staphylococcus Broth
Enriched media containing 10% NaCl, which selects for Staphylococcus since Staphylococcus prefer
o
the higher salt concentration, which inhibits most other organisms
Endo Agar
Selective for Gram-negative. Combination of sodium sulfite and the basic fuchsin inhibits Gramo
positive bacteria.
Differential for lactose fermentation.
o
Basic fuchsin acts as indicator of acid production from lactose fermentation.
26.
27.
28.
29.
30.
13
Opt
5C
30-37C
55-65C
Max
15-20C
45C
70-90C
pH:
Group terms are based on pH in growth environment.
Acidophiles: require an acidic pH growth range.
Neutrophiles: best growth at pH 6.5 to 7.5.
Alkylophiles/basophiles: require alkaline growth conditions or growth best above pH 7.0.
Oxidation-Reduction Potential:
Oxidation-reduction (OR) potential refers to the presence or absence of oxygen. Oxygen-rich environments remove
net electrons (aerobic), while oxygen-deficient environment have a net increase in electrons (anaerobic). Microbes
require the presence of oxygen, the absence of oxygen, or tolerate both conditions (facultative). In determining this
trait the reducing agent thioglycollate can remove oxygen, except at the top/meniscus. Thus, you can determine the
position of growth in the tube and group the bacteria accordingly. Resazurin is used as a redox indicator: colorless in
reducing conditions and pink when oxidized.
Group terms are based on OR requirements or tolerance.
Strict aerobes: requires oxygen for metabolism and maintenance of cell structure. Bacteria grow only at
the top of the tube.
Facultative species: can grow with or without oxygen. Bacteria grow throughout the tube.
Strict anaerobes: inactivated by oxygen exposure (metabolism is fermentative), cell structure may be
altered by oxygen. Bacteria grow only at the bottom of the tube.
Osmotic Pressure (aw):
The osmotic pressure is produced by an imbalance of free unbound water on either side of the semi-permeable cell
membrane. High solute (salt) concentration (hypertonic) binds water and intracellular water diffuse out to inactivate
the cell. Some species can control the osmotic pressure changes and tolerate the hypertonic state. There are species
that require Na+ ions for stability and require salt in their environment. This test uses NaCl to reduce free water
availability (not bound by solutes).
Group terms are based on the salt concentration required:
Halophile: require higher concentrations of salt to survive and grow. From about 5% to 30% (w/v) salt.
Halotolerant: do not require higher salt concentrations but can tolerate it.
Non-halophile: grow best with 0.5% salt (if that solute is used). Other solutes bind water and their
concentration is important.
Osmophile: tolerates osmotic pressure produced when other solutes (besides salt) are in high concentration
in their growth environment.
Ultraviolet (UV) Irradiation:
Unlike temperature, pH, osmotic pressure and OR potential, UV irradiation is not in every environment. DNA in the
cell absorbs at 260nm (UV wavelength) maximally, causing damage (especially thymine dimers). Tolerant species
can correct the damage.
Group terms:
UVR: ultraviolet resistant.
UVS: ultraviolet sensitive and lethal.
14
15
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.
Streptococcus:
o Using the Blood Agar plates from last lab identify three alpha- or beta-hemolytic
Streptococcus by identifying the chain formation of the Gram-positive cocci.
Inoculate each of these three colonies into a Nitrate Broth and perform a catalase test
on each (record these results as well as the hemolytic results from the Blood Agar)
Into the 4th Nitrate Broth inoculate with the provided Streptococcus culture.
16
17
Perform isolation streaks of the Salmonella containing mixture onto each of the
selective/differential media provided.
From the five selective/differential plates select seven isolated colonies that are presumptive
for being Salmonella based on their appearance and inoculate each into a SIM, Urea, and KIA
media.
o Colonies to be selected:
EMB: lactose-negative bacteria (colonies do not change color)
DES Citrate: lactose-negative bacteria (colonies do not change color)
BGA: lactose-negative (colonies appear pink/white surrounded by red media)
SS Agar: black colonies
BSA: black colonies
Day 2:
Day 3:
18
19
MYCOLOGY
Terminology:
Mycology is the study of fungi (p) (fungus, s).
Myco- or -mycete indicates fungi or similarity to fungi.
Cellular Traits of Fungi:
They are nonmotile eukaryotes that possess nuclei and organelles.
Fungi seem plant-like but are not plants; they do not possess chloroplasts and are not
photosynthetic.
Large cells ( 10m), making them visible using low magnification (10X objective).
Contain a simple cell wall of chitin (polysaccharide), not similar to the complex peptidoglycan of
bacterial cells, or cellulose cell wall of plants.
Fungi are absorptive heterotrophs, they release exoenzymes into the environment and then
absorb and digest the nutrients.
Most are saprophytes that decompose dead and decaying organic matter; few are parasites of
plants, animals and humans.
Most fungi are aerobic; few are facultative or aerotolerant anaerobes.
Informally divided into:
a. Filamentous molds that create multicellular hyphae. It also includes macrofungi which
produce fleshy reproductive structures (puffballs, mushrooms, and shelf fungi).
b. Unicellular yeast that reproduce by budding.
c. Dimorphic fungi which have both mold (25C) and yeast (37C) life-cycle stages.
Cultural Traits of Fungi:
Fungi tolerate a broad range of pH and osmotic pressures.
Have a narrower tolerance of temperature and redox conditions.
Media selective for fungi will have an acidic pH between 4.8 to 5.0.
Able to metabolize complex substrates, including woody wastes such as cellulose, lignin, tannins,
and hemicelluloses.
o Fungi are critical for decomposition of forest litter and dead trees (saprophytes).
o Yeast form bacteria-like colonies.
Fungal Infections:
Fungal infections are called mycoses (p)/mycosis (s).
Superficial mycosis: topical (skin, hair, nails) infection.
Cutaneous mycosis: infection of the dermis.
o Tinea infections (ringworm, jock itch)
o Candida infections (vaginitis, thrush)
o Subcutaneous mycosis
o Deep seated/systemic mycosis includes respiratory or deep tissue infections (very serious
conditions).
o Coccidiomycosis
o Aspergillosis
Properties of Unicellular Fungi (yeast):
Grow as solitary cells and reproduce by budding.
Distinguished according to the presence or lack of a capsule, size and shape of the cell and method
of formation of the daughter cell.
20
Zygomycetes
Vegetative hyphae filaments are branched and nonseptate.
Zygote undergoes meiosis and produce a sexual zygospores.
Can also produce sporangiospores (asexual spores) in a sporangium located on the apical end of
the hyphae.
Includes Mucor and Rhizopus spp.
II. Ascomycetes
Contain septate hyphae.
Produce a haploid ascospores through meiosis in a sac called ascus after mating.
May fuse to form another generation of diploid vegetative cells.
Can also produce asexual conida.
a) Single-cell yeasts:
o Reproduce asexually by binary fission and budding.
o Reproduce sexually by mating. Cell is now an ascus containing many small ascospores.
o Yeasts often produce pseudohyphae by repeated, sequential budding to produce a
filament-like extension of the cell.
o Includes Sacchromyces cervisiae and Schizosaccaromyces octosporus.
b) Multi-cell filamentous molds:
o Sexual mating types.
o Septate vegetative hyphae.
o Asexual conidiospores in chains.
o Includes Penicillium and Asperigillus.
21
III. Basidiomycetes
Septate hyphae that are compacted into large fruiting bodies (mushrooms, shelf fungi, puffballs,
etc).
IV. Deuteromycetes
Also known as fungi imperfecti.
Nearly indistinguishable from septate, conidiospore-forming ascomycetes.
This group is has never been scientifically proven to have a sexual reproductive stage.
Sexual reproduction in this phyla is still considered possible, just unproven.
22
23
ANTISEPTIC EVALUATION
1.
2.
3.
Tube #
Broth
Dilution
Antiseptic
DI Water
Organism
1
5.0 mL
1:10
1.0 mL
3.0mL
1.0mL
2
5.0 mL
1:25
1.0 mL
3.0mL
1.0mL
3
5.0 mL
1:50
1.0 mL
3.0mL
1.0mL
4
5.0 mL
1:75
1.0 mL
3.0mL
1.0mL
5
5.0 mL
1:100
1.0 mL
3.0mL
1.0mL
6
5.0 mL
1:10
1.0 mL
4.0mL
-----
7
5.0 mL
--------4.0mL
1.0mL
8
5.0 mL
--------5.0mL
-----
9
5.0 mL
-----------------
24
UNKNOWNS
Your unknown will contain 2 different organisms from the following list:
Acinetobacter
Alcaligenes
Bacillus
Corynebacterium
Enterobacter
Eschericia
Klebsiella
Lactobacillus
Micrococcus
Moraxella
Mycobacterium
Neisseria
Proteus
Pseudomonas
Salmonella
Shigella
Staphylococcus
Streptococcus
Procedure:
1. Record the number of your unknown
2. Gram-stain your unknown.
Record Grams result, shape, and arrangement of the organisms in your unknown.
3. Streak your unknown mixture onto 2 TSA plates, and 2 BHI plates. Place 1 TSA and BHI plate in
the incubator, and the other set of plates at room temperature.
4. Identify isolated colonies that appear different on the isolation streaks.
Gram-stain to ensure isolation and identification.
5. Transfer each different isolated colony onto 2 TSA and 2 BHI slants.
1 TSA and 1 BHI slant will be your working stock, while 1 TSA and 1 BHI will be your
reserve stock.
When you use a slant for inoculation, discard that slant and reinoculate a fresh slant from
the reserve stock. The old reserve stock will be come your working stock for the next lab,
and the newly inoculated slant will become the reserve stock.
6. Using the following flowcharts, all other materials provided throughout this class, and Bergeys
Manual (Reference Section of the Library) determine the identity of your unknowns.
7. Submit your results using the dichotomous key on Canvas. Include your unknown number. Circle
the organisms you find and then (using a highlighter) highlight the path you took to figure out
your organisms.
Common Errors When Working with Unknowns:
1. Old cultures (older than 18-24 hours) often produce gram variable reactions. It is best to Gram
stain young cultures.
2. Cultures that have not been properly isolated often produce results that do not fit any particular
pattern. To check for purity, Gram stain and/or re-streak for proper isolation.
3. Contamination with outside bacteria is fairly common, remember to use proper aseptic techniques
and rotate your bacterial stock accordingly.
4. Spore stains are best performed on older cultures as spores will not be formed until your organism
is stressed.
5. Oxidase test is best performed on fresh cultures as older organisms can tend to give false negative
results.
25
26
OBJECTIVE
SIGNIFICANT
COMPONENT
S
Phenylethanol
(PEA) inhibits
Gram-negative
growth
MEDIUM
TYP
E
Phenylethanol
Agar (PEA)
Sele
ctive
Eosin Methylene
Blue Agar (EMB)
Sele
ctive
&
Diffe
renti
al
Desoxycholate
Agar (DES)
Sele
ctive
&
Diffe
renti
al
Blood Agar
Starch Agar
INDICATOR
RESULTS
Growth = Gram-positive
No growth = Gram-negative
Lactose, eosin,
and methylene
blue dye
Lactose, and
desoxycholate
(detergent)
Diffe
renti
al
Detects hemolysins by
observing hemolysis
5% sheep red
blood cells
(RBC)
Action on RBC
Diffe
renti
al
Soluble starch
Lipase Agar
Diffe
renti
al
Milk Agar
Diffe
renti
al
Milk (containing
opaque casein)
Gelatin
Diffe
renti
al
Gelatin
Resolidification of gelatin at
lower temperature
Tryptone Broth
Diffe
renti
al
Detects tryptophanase
which hydrolyzes
tryptone to indole Tests
for presence of indole
Tryptone
polypeptide
containing
tryptophan
Nitrate Broth
Diffe
renti
al
Sodium nitrate
Motility Media
Diffe
renti
al
Determines if the
bacteria is motile
Tetrazolium
chloride
(growth
indicator)
Tetrazolium chloride:
red=bacteria growth
Diffe
renti
al
Detects moderate/heavy
acid from lactose
fermentation, presence of
caseinase and reductase
Lactose,
Litmus, casein
from milk
**(1) moderate acid production from lactose = pink, liquid in tube; (2) heavy acid production from lactose = pink, hard or soft curds (coagulation of casein by acid);
(3) alkaline products from metabolism = blue, liquid; (4) reduction of litmus dye to colorless in lower half of broth = viewed as whitish from natural milk color when the litmus
dye is no longer in the oxidized colored state. Due to bacterial reductase in milk; (5) loss of opaque casein protein by caseinase = proteolysis (peptonization),
usually seen starting at the top and increases downward; (6) slimy strands suspended in liquid state of medium = ropiness trait produced by capsule-producing bacteria
MEDIUM
Urea Broth
TYPE
OBJECTIVE
Phenylalanine
Oxidase
Catalase
INDICATOR
Phenol red: yellow=acid,
red=neutral, cerise=alkaline
Methyl red is added after growth
(3-5 drops), a pH below 5.1=red
VP I & II are added after growth
(10 drops each)
Bromothymol blue: yellow=acid,
green=neutral, blue=alkaline
Differential
Methyl Red
(MR)
VogesDifferential Detect 2,3-butanediol fermenters
Proskauer (VP)
Simmons
Detect use of citrate as sole
Differential
Citrate Slant
carbon source
Phenol Red
Detects fermentation of selected
(PR) Sugar
Differential sugar
Fermentation
Detects production of gas
Kligler's Iron
(KIA) Tube
SIGNIFICANT COMPONENTS
Glucose
Citrate
Sugar (variable) and Durham
tube
RESULTS
Red to cerise = urease (+)
Yellow to light orange = urease (-)
Dye stays red = mixed acid fermenter (+)
Turns yellow = mixed acid fermenter (-)
Red on top = 2,3-butanediol fermenter (+)
No red color = 2,3-butanediol fermenter (-)
Growth on slant and blue color = (+)
No growth = (-)
Yellow = fermenter (+)
Light orange to red = fermenter (-)
Gas in tube = gas production (+)
Red slant/yellow butt = glucose fermenter
All yellow = glucose & lactose fermenter
Black = H2S production
Green = phenylalanase (+)
No color change of ferric chloride =
phenylalanase (-)
Dark red/purple to black = oxidase (+)
Remain colorless = oxidase (-)
Bubbles = catalase (+)
No bubbles = catalase (-)
CELLULAR STAINING
STAINING
TYPE
Negative
Simple
Simple
Simple
Gram
Differential
Acid Fast
Differential
Spore
Structural
PRIMARY
STAIN
Indian ink or
Nigrosin
Methylene
blue
Crystal violet
Carbol
fuchsin
Malachite
green
MORDANT
DECOLORIZE
R
SECONDARY
STAIN
RESULTS
Background stains black, bacteria are
transparent
All bacteria stain blue
Iodine
Ethyl-alcohol
Safranin
(Heat)
Acid-alcohol
Methylene blue
(Heat)
Water
Safranin