EXERCISE 6: Pure Culture Techniques
Colony
- A visible mass of microorganisms growing on an agar
surface arising from a single spore or vegetative cell or from
a group of the same microorganism attached to one
another in clumps or chains
- Often have distinctive appearance that distinguishes one
microbe from another
Pure Culture
- A culture wherein all cells are of the same origin or
descendants of the same cell
- A laboratory culture containing a single species of organism
derived from a single cell
Axenic Culture
- A culture of organisms from a single species growing in a
medium entirely free of other organisms
- Literally without strangers. A free system in which all
biological populations are defined, such as a pure culture
Mixed Culture
- contains more than one species of microorganism
Normal Flora/Microbiota
- Mixture of organisms naturally found at a specific site
- Bacteria which are found in or on a host on a semi-
permanent basis without causing disease
Contaminant
- Any material or organism that is not supposed to be present
in a certain place or object
Lyophilization
- A type of long-term preservation of a culture
- A bacterial culture is suspended in a protective medium
which are dehydrated while in a frozen condition and
sealed under a vacuum
DIRECT METHOD
Micromanipulator Method
- Used with a microscope to pick a single cell from a hanging
drop preparation of a mixed culture
Several hanging drops of a diluted culture are placed on
a special sterile coverslip using a micropipette
Using the microscope, a hanging drop containing a single
microorganism cell is searched
This cell is drawn into the micropipette using a gentle
suction and then transferred to a large drop of sterile
medium
Once the cell increases in number, the drop is transferred
into a suitable culture tube
INDIRECT METHODS
Streak-Plate Method
Repeated picking and re-streaking of a well-isolated
colony until a pure culture is obtained which can be
transferred into a final medium
For organisms that grow well on an agar plate. Useful
when microorganism to be isolated is present in large
numbers compared to the other microorganisms. As the
pattern is traced, bacterial cells are rubbed off the
loop/needle and over the medium in paths of fewer and
fewer cells.
Goal: To produce good spacing between colonies so
separate colonies can be seen by the naked eye
Agar is the solidifying agent of the medium that becomes
liquid when boiled and solidifies at around 42 deg.C
METHOD:
Cool down the melted agar to 45-50oC before pouring to
avoid condensation of moisture on the petri dish cover.
Moisture on the cover can fall down on the colonies so
the organism of one colony can spread to other colonies
Never streak a plate before the medium is firm
Only obtain a loopful of bacteria culture once. Do not get
another loopful for the following streaks because it
defeats the purpose of dilution technique
Do the streaking gently to avoid damaging the medium
Flame the loop in between streaks to further promote
dilution of the microbe concentration. Allow it to cool
before streaking again because extremely high temp can
damage the microbe
Streak the next pattern by passing over a portion of the
previous streak
Continue flaming then streaking until the pattern is
completed
Incubate the petri dish in an inverted position to prevent
condensation from falling onto the agar surface
Always heat the opening of petri dish before and after
each streak
Spread the organism as thinly as possible
Isolated colonies are located at the end of each streak
STREAKING PATTERNS
1. Quadrant Streak
Red: 5 to 6 streaks
Blue: 6 to 7 streaks
Green: As many as possible
Flame the loop in between streaks. Cool the
loop by touching an area of the sterile media
for 5 seconds then continue streaking
2. T-Streak
three-section streak
Turn plate 90 degrees and drag the loop through
the first streak for two to three times then
continue to do zig zag lines without touching the
previously streaked area
Flame the loop in between streaks
3. Radiant Streak
Black: Deposit of bacteria
Red: 7 to 8 streaks
 4. Continuous Streak Limitations desired microbe microbes: not heat-sensitive;
should be in can withstand the T of melted
large quantities NA (45-50oC)
higher subsurface colonies may not
chance of it be useful when using
being isolated differential media; only the
surface colonies might be
After black, rotate the petri dish 180 deg C. DO observed
not flame the loop. Continue streaking half the Description of Colonies
plate red. Size
Pour-Plate Method o Measure using a millimeter ruler
Dilution of the original bacterial sample several times o Average of similar sized colonies
which reduces the microbial population sufficiently to Color
obtain individual colonies upon plating o Non-chromogenic: no pigment/white
Also known as the loop dilution method o Chromogenic: give color
The magnitude of the microbial population is not known Opacity
beforehand so it is necessary to make several dilutions to o Opaque, translucent, transparent
obtain at least one plate with distinctly separated Elevation
colonies in an agar medium.
Dilution can be done by transferring a small volume or a
loopful of solution from the first tube to the next fluid
agar media.
Margin

Configuration

Melt NA in a water bath. Maintain the T bet 45-50oC

during dilution. Do not allow NA to solidify in the tube.
Transfer 1 loopful of bacterial suspension into a tube of
sterile saline.
Rotate the saline tube between palms for 10 times to
ensure uniform distribution of bacteria.
Transfer 2 loopfuls of saline tube to a tube of melted
nutrient agar = tube 1. Mix contents of tube 1.
Transfer 2 loopfuls of tube 1 into another tube of melted
nutrient agar = tube 2.
Pour contents of tube 1 into a petri dish = petri dish 1.
Rotate tube 2 then transfer 2 loopfuls from tube 2 to
another tube of melted nutrient agar = tube 3.
Pour contents of tube 2 into a petri dish = petri dish 2.
Rotate tube 3 then transfer its contents into a petri dish
= petri dish 3.
Pour contents of tube 4 containing uninoculated sterile
nutrient agar into a petri dish = petri dish 4 (CONTROL).
Incubate all plates in an inverted position at 35 deg C for
48 hours.
Streak-Plate Pour-Plate
Skills HIGH: streaking LOW: dilution does not
needed requires skill
Materials LESS MORE: because of the several
dilutions and plating
Subculturing Techniques
Process of transferring some microbial cells from a
previous culture into a fresh growth medium
Used to prolong the life of the isolated colony and/or to
increase the number of cells of the isolated colony
Usually done after developing a pure culture using the
various pure culture techniques
Short-term method of preserving culture
METHOD
Obtain a colony from the petri dish then transfer to a
suitable fresh broth or slant.
The subculture will be incubated for at least 24 hours to
allow the cells to increase in number
The subculture will be stained to determine if a pure
culture is obtained.
Ways to preserve a pure culture
Short term: periodic transfer to a fresh medium
Long term: lyophilization
 EXERCISE 7: Anaerobic Culture Methods
Effects of Different Enzymes on Oxygen
toxic forms of O2: superoxide and hydroxyl radical
several mechanisms to eliminate these toxic O2 forms
o superoxide dismutase superoxide O2 + H2O2
(removes the extra electron on one superoxide then
places it on another superoxide = one is electron less
which becomes O2; the one with an extra electron
picks up 2 H+ resulting to H2O2)
o Since H2O2 is also toxic, another enzyme is used to
detoxify it. Catalase converts H2O2 into H2O and O2.
o Peroxidase can also convert H2O2 into water in the
presence of a coenzyme such as NADH2.
not all organisms have these enzymes or mechanisms
Types of Bacteria Based on O2 Requirements
Obligate aerobes grow in the presence of O2; can
neutralize toxic forms of O2 through the enzyme systems
SOD and catalase
Facultative anaerobe aerobic and anaerobic; can
neutralize toxic forms of O2 but can still grow even
without O2
Obligate anaerobe can only grow in environment
without any O2; cannot neutralize toxic forms of O2
Aerotolerant anaerobes cannot use O2 for growth but
has SOD not harmed by O2
Microaerophiles grows in the presence of low
concentration of O2 (~5% O2)
Anaerobic Culture Methods
common principle: provide an environment where dissolved/free
O2 is depleted and eventually removed to allow anaerobes to grow
1. Wrights Tube Method
Named after James H. Wright
Pyrogallol: reducing agent
o Activated by NaOH
o Removes free O2 in the tube
Messy
Difficult to recover organism inside the tube
Wetness inside the tube creates difficulty
METHOD
Inoculate the surface of the agar slant
Flame the cotton tip Burnt cotton consumes O2 gas
Using the butt of a needle or loop, push the burnt cotton
until it nearly touches the end of the slant.
Place pyrogallic acid crystals above the cotton stopper
Add around 2 mL NaOH NaOH oxidizes pyrogallol and
removes the free O2
Place a cork stopper to close the tube
Invert immediately
Seal the tube by dipping the stoppered end into melted
paraffin
2. TGYA Shake
aka shake tube
supports the growth of a broad spectrum of microbes
standard medium for the bacteriological plate count of
milk and dairy products
Medium is inoculated when liquefied, shaken to disperse
the microbes, and solidified
O2 requirement: based on location of growth
o top: require high amounts of O2
o bottom: harmed by O2
o middle: may not be affected by O2 or does not need
high amounts for growth
Tryptone: source of amino acids
Glucose: source of energy
Yeast: rich in vitamins, minerals and digested nucleic acid
Agar: solidifying agent, can suspend microbes
3. Thioglycolate Broth Tubes
Supports the growth of aerobes, anaerobes, and
microaerophiles
Contains glucose and cystine
NA thioglycolate: reducing agent
o reduces redox potential of media
o its free sulfhydryl groups reduce free O2 which
creates the anaerobic environment
Agar: favors growth of anaerobes at the bottom
Resazurin, MB: redox sensitive dyes used as indicators for
the presence of oxygen
O2 Resazurin Methylene Blue
(+) pink Blue
(-) colorless Colorless
Upper: colored; middle and bottom: yellow to colorless
DO NOT shake tube to avoid oxidizing the broth
If more than 1/3 of the broth appears reddish, the tube
should be reheated in a water bath in order to drive off
the O2 before use
4. Brewers Petri Dish Method
Named after John H. Brewer
 Brewers special cover: fits on a normal petri plate 6. Oxyrase
bottom in such a way that its circular ridge rests on the Respiratory enzyme derived from bacteria
agar, thereby protecting most of the surface from the Reduces O2 to H2O
exposure to O2 Eliminates complications and expense of bags, jars,
Brewers anaerobic agar: high concentration of anaerobic incubators, and chambers
thioglycolic acid which reduces any O2 present Oxyplates contain a pre-poured media with oxyrase
anerobic environment Can be opened and closed several times and it will
regenerate anaerobic conditions
Can be placed in a standard incubator
7. Candle Jar
5. Anaerobic Jar Large sealed jar containing a lighted candle which
GasPak Anaerobic System consumes oxygen
o H2: generated to remove O2 by forming H2O Elevates CO2 inside the jar
o CO2: generated for the growth of fastidious bacteria
Enhances growth of microaerophiles
(bacteria with complex nutritional requirement)
candle light turns off = no more O2
H2 & CO2 are generated when H2O is added to the plastic
8. Vacuum and Gas Displacement
envelope/GasPak which contains NaHCO3 and NaBH4
Air is pumped out of the jar
Palladium pellets: catalyzes the reaction between H2 & O2
O2 is displaced with inert gases like N2 and CO2
Methylene blue: indicator for the presence of O 2
Already replaced by the GasPak method
Description of Growth
Amount Surface Subsurface Sedimentation
None Ring Turbid Granular
Slight Pellicle Granular Flocculent
Moderate Flocculent Flocculent Flaky
Abundant Membranous Flaky Viscid

LECTURE 7: Mycology
Characteristics of Fungi Yeasts
Fungi Bacteria Unicellular; Filamentous
Cell type Eukaryotic Prokaryotic Typically oval/spherical
CW Glucans (cellulose), Peptidoglycan Candida albicans: oral thrush, vaginal candidiasis
Mannans, Chitin Saccharomyces cerevisiae: aka brewers/bakers yeast
CM Sterols: ergosterol Sterols absent, except in Molds
Mycoplasma Multicellular; Filamentous
Spores Reproductive spores Endospores (non- Typically oval/spherical
(sexual/asexual) reproductive) Mildew commonly seen in plants & household surfaces
detach from the parent for survival in adverse Rusts not iron oxides, but plant fungi that appear as a
and germinates into a conditions powdery, yellow-orange substance (thus look like rust)
new mold Smuts attack grasses, wheats, corn
Metabolism Limited to heterotrophic Heterotrophic/ Consist of hyphae which are multicellular filaments
(can metabolize complex autotrophic o VEGETATIVE: non-reproductive; spread across food
carbohydrates) source; obtain nutrients
pH 3.8-5.6 (acidophiles) 6.5-7.5 o AERIAL: reproductive; grow vertically; produce spores
T (oC) 22-33 (saprophytic) 20-37 (mesophiles) o SEPTATE: presence of septa between cells; has pores
30-37 (parasites) that allow flow of cytoplasm and nutrients; septa can
O2 strictly aerobic (molds) Aerobic toanaerobic completely close to prevent damage of the rest of the
facultative anaerobe filament
(yeasts) o COENOCYTIC: form one long cell with several nuclei
Light Non-photosynthetic some are photosynthetic o Allow nutrients to move quickly
Sugar 4.5-5.0% 0.5-1.0% o Entire filament can die if ruptured
Antibiotic Resistant to penicillins, opposite Fleshy Fungi
susceptibility tetracyclines, Multicellular; Filamentous
chloramphenicol Produce a thick reproductive body
Sensitive to griseofulvin Mushroom, Puffballs, Coral Fungi
Dimorphic Fungi
2 forms of growth
CO2 concentration-dependent
Temperature-dependent
o 37oC: YEAST-LIKE
o 25oC: MOLD-LIKE
Conversion to yeast form of these pathogenic fungi at
body temperature plays a role in its pathogenesis
Blastomyces dermatitidis: BLASTOMYCOSIS
Histoplasma capsulatum: HISTOPLASMOSIS
Coccidioides immitis: COCCIDIOIDOMYCOSIS
Paracoccidioides brasiliensis: PARACOCCIDIOIDOMYCOSIS
Sporothrix schenckii: SPOROTRICHOSIS
Life Cyle of Fungi
Fungi can reproduce both asexually and sexually.
The life cycle of a fungi may lack one reproductive stage
i.e., it only produces either asexually or sexually.
Most fungi have both reproductive stages in their life
cycle.
ASEXUAL REPRODUCTION
Produces genetically identical fungi through
o Fragmentation of hyphae (fragments of hyphae can
grow into new colonies)
o Budding
o Production of spores
Conidiospores released from the side or tip of
the hyphae
Sporangiospores produced and release from a
sac called sporangium
asexual spores are produced by one parent only
through mitosis
Spores allow fungi to expand their distribution
and colonize new environments.
EXERCISE 8: Methods of Culturing Fungi
Media for Culturing Fungi
1. Saborauds Dextrose Agar
Created by and named after Raymond Saboraud
Most frequently used simple medium
4% glucose, 1% peptone, 2% agar
o peptone contains enzymatic digest of casein and
animal tissue which provide a nutritious source of
amino acids and nitrogenous compounds for the
growth of fungi and yeasts
SEXUAL REPRODUCTION
Produces a fungus with combined genetic traits
Formed from 2 organisms
Phases:
o PLASMOGAMY: 2 haploid cells fuse and coexist in a
single cell
o KARYOGAMY: 2 haploid nuclei fuse and form a
diploid zygote nucleus
o MEIOSIS: diploid nucleus produces haploid nuclei
(sexual spores)
Medically Important Fungi
1. Zygomycota (Conjugation Fungi)
Coenocytic hyphae
E.g. saprophytic molds (Rhizopus stolonifera, common
black bread mold)
Sexual spore: ZYGOSPORES
o Large spores enclosed in a thick wall
o Fusion of morphologically similar nuclei
Asexual spore: SPORANGIOSPORES
2. Ascomycota (Sac Fungi)
Spores freely detach from the chain at the slightest
disturbance and float in the air like dust
E.g. molds with septate hyphae, some yeasts
Sexual spore: ASCOSPORES
o Fusion of morphologically similar or dissimilar nuclei
o Produced in a saclike structure called ascus
Asexual spore: CONIDIOSPORE
3. Basidiomycota (Club Fungi)
Produces large fruitbodies like mushroom and puffball
E.g. molds with septate hyphae, fleshy fungi
Sexual spore: BASIDIOSPORES
o Formed externally on a club-shaped pedestal called
a basidium
o Usually 4 basidiospores per basidium
Asexual spores: CONIDIOSPORES
4. Deuteromycota
Fungi imperfecti
holding category for unclassified fungi
Fungi whose sexual cycle had not been observed
TELEOMORPH: reproduce sexually and asexually
ANAMORPH: lose ability to reproduce sexually
5. Microsporidia
Lack mitochondria and microtubules
Obligate intracellular parasites
Sexual reproduction is not observed (probably occurs
within the host)
Reported cause of several human diseases
o Chronic diarrhea
o Keratoconjunctivitis, most notably in AIDS patients
o Dextrose is the fermentable carbohydrate
incorporated in high concentration as a carbon and
energy source.
o Agar is the solidifying agent.
pH 5.6 to inhibit bacterial growth
2. Emmons Medium
Adjusted by C. Emmons
2% glucose
 40mg/L chloramphenicol or tetracycline to inhibit a wide
range of gram-(+) and gram-(-) bacteria
Gentamicin is added to further inhibit gram-(-) bacteria
Description Method
SLIDE CULTURE 1. Take a clean and sterilized glass slide.
Moist Chamber Method 2. Melt a tube of Sabourauds agar in
water bath and cool to 45C.
3. Inoculate cooled melted medium with
spores of the fungal culture. Shake to
distribute spores.
4. Transfer small amount of inoculated
medium on the surface of the glass permits fungi to be studied hard to maintain sterility in
slide with a sterile pipet and spread in
a thin layer.
5. Place the slide in a moist culture no need to remove a portion
chamber
- 2-3 layers of filter paper below petri
dish (moistened)
- U-shaped glass tubing (stage)
6. Incubate the fungal culture at room T.
DO NOT INVERT.
7. Examine under a microscope.
COVER GLASS 1. Melt a tube of Sabourauds agar in allows ease in assessing the arrangement of spores may be
Extensively used with a water bath. Cool to 45C and
bacteria and fungi inoculate heavily with spores from
Some fungi tend to the fungal culture. Rotate between
disintegrate as soon as palms to distribute the spores.
mounted 2. Transfer 1-2 loopfuls of inoculated
Cladosporium, Monilia, agar on a flamed, degreased cover
Alternaria have spores glass.
connected in very fragile 3. Seal the brim of the glass slide with
chains that can fall apart white petrolatum and invert the
even in the slightest inoculated cover glass over the
movement of air depression.
Mounts of these fungi 4. Incubate the preparation at room
invariably reveal only T. DO NOT INVERT.
loose spores and a 5. Examine under a microscope and
network of hyphae record the results
GIANT CULTURE 1. Inoculate the center of a macroscopic characteristics of wet mount slides do not
Viewed by making wet Sabourauds agar plate with a
mount slides small amount of the fungal
culture.
2. Incubate the plate at room T.
SCOTCH TAPE 1. Touch the adhesive side of the hyphae,
To examine fungi in their transparent tape to the surface of
natural state fungal colony
2. Place a drop of lactophenol cotton
Avoids disruption of the
blue or aniline blue with a clean
characteristic microscope slide.
arrangement of spores 3. Stick the tape over the surface of the
during observation slide containing the lactophenol
Use of lactophenol cotton blue drop
cotton blue with KOH to 4. Examine under the microscope.
enhance visibility of the 5. Note the characteristic shape and
fungi arrangement of spores and other
structures.
Rhizopus sp. Aspergillus sp. Penicillium sp.
pH 6.8-7.0: seems to enhance the growth of some
pathogenic fungi, such as dermatophytes
Advantages Disadvantages
hyphae, sporangiophores, arrangement of spores may be
spores remain more or less disrupted when pressure is
intact when stained applied to the coverslip
a rapid method of preparing rapidly exhausts oxygen and
fungal colonies for nutrient supply; quick to turn
examination and identification acidic
virtually in situ with as little prolonged periods
disturbance as possible allows only a small amount of
sample to be cultured
of the fungus from a culture
plate and transfer it to the
slide
less chance for the features
that are key to identification
to be damaged
culture through direct disrupted when pressure is
observation under the applied to the coverslip
microscope
the fungal colony can be usually reveal the
noted arrangement of spores
hyphae & sporangiophores
break up when transferring
hyphae to slide
sporangiophores, arrangement of spores may be
spores remain intact when disrupted when pressure is
stained applied to the coverslip
less safe to handle because
the sample is taken from giant
culture method
only the superficial structures
of the fungi tend to stick to the
tape.
troublesome (difficult) and
awkward (uncomfortable) in
inexperienced hands
Trichophyton sp.
 EXERCISE 9: Biochemical Characteristics
Biochemical Reactions
catalyzed by enzymes which are produced by individual
genes that are specific for a species
A microorganism can be identified through the results of
biochemical tests and its morphological
characterizations.
Biochemical tests use biochemical reactions that are
specific for a species.
Enzymatic Reactions in Bacteria Lipid Hydrolysis
Respiration: oxidative bacteria
o Bacteria that utilize glucose in the presence of
oxygen (respiration)
o Use organic compounds as electron donors with
oxygen as the ultimate electron acceptor
o Products during release of energy: CO2and H2O
o Respiration is accomplished by the cytochrome
enzyme system
Fermentation: fermentative bacteria
o Use organic compounds for energy: act as electron
donor and acceptor because there is no oxygen
o Lack the cytochrome enzyme system
o Complex end-products
CO2, H2O, acids, aldehydes, alcohols, H2, CH4
Oxidizable and reducable
Facultative bacteria: both oxidative and fermentative
Streaking the Plates
Divide the outside bottom half of petri dish into 2 (paper
nalang) Fermentation of Carbohydrates
Pour the three media into separate petri dishes
Allow solidification of medium.
Streak each microbe on the surface
Incubate at 35 deg.C for 48-72 hrs
For starch hydrolysis, flood the surface with grams iodine
solution
Starch Hydrolysis
Medium: STARCH AGAR PLATE
Indicator: GRAMS IODINE SOLUTION
(+): CLEAR AREAS next to the growth
o When iodine comes in contact with a medium
containing starch, it turns blue. If starch is
hydrolyzed and starch is no longer present, the
medium will have a clear zone next to the growth.
Starch is a large molecule the has 2 constituents:
o amylose (straight chain polymer of 200-300 glucose
units): causes the blue color of starch with iodine
o amylopectin (larger, branched polymer with
phosphate groups): reddish-brown color of starch
with iodine
Casein is the predominant protein in milk. Its presence
causes milk to have its characteristic white appearance.
Medium TRIBUTYRIN AGAR SPIRIT BLUE AGAR
Indicator tributyrin agar spirit blue agar
(+) CLEARING of the CLEAR HALO around the
medium growth
This ability of bacteria to decompose fats plays a role in
the rancidity of certain foods, such as margarine.
Tributyrin: Opacity is due to fat.
Spirit blue: Spirit blue agar contains an emulsion of olive
oil and spirit blue dye.
Clearing, not lightening of medium, is the positive VR
Fermentation results in by-products such as lactic acid,
alcohol, CO2, H2
o Acid: Ethanol, acetic acid, lactic acid, formic acid,
succinic acid, propionic acid, citric acid, butyric acid
o Gas: carbon dioxide, methane, hydrogen gas
o Others: acetone, butanol, glycerol, sorbose (from Vit
C), isopropyl alcohol, acetoin, butanediol
Presence of acid is detected by color change
Presence of gas is detected by the formation of a void in
an inverted Durham tube
Fermentation tubes
o Nutrient broth
o 0.5% carbohydrate
sugar (glucose, lactose, sucrose, maltose,
mannitol)
indicator
peptone (protein derivative that can be
metabolized when there is no more available
nutrient basic by-products) or yeast extract
o Phenol red (pH 6.8-8.2)
pH > 7: RED
pH < 7: YELLOW
o Durham tube

Protein Hydrolysis / Proteolysis / Peptonization

Medium: MILK AGAR PLATE
Indicator: MILK AGAR
(+): CLEAR AREAS next to the growth
 METHOD
Inoculate the organism in 5 different carbohydrate
broths with one loopful.
Tilt the test tube then slowly allow the durham tube to
slide down. Avoid having bubbles in the durham tubes
Create a control tube for each carbohydrate broth
Incubate at 35OC for 45 hrs Triple Sugar Iron Agar Results
RESULTS

AG acid and gas (yellow with bubbles)

A acid (yellow without bubbles)
a slightly acidic (orange)
B basic (pinkish-red to pink)
O carbohydrate not fermented (same with control)
Hydrolysis of Gelatin
Gelatinase: enzyme that can hydrolyze gelatin
Inoculate the nutrient gelatin tube by a stab down to the
butt technique using a needle. Production of Indole
Incubate at 34oC for 48 hrs bacterial enzyme tryptophanase breaks down
o (-): refrigerate for another 4 to 5 days since some tryptophan into indole and pyruvic acid
organisms produce gelatinase at a very slow rate
o (+): LIQUEFACTION
Once the medium lacks gelatin, the firm
characteristics are lost -> liquefaction
Hydrogen Sulfide Production
Example: E. coli
H2S is produced by bacteria through:
DETCECTION OF INDOLE
o Breakdown of S-containing amino acids
Indole reacts with the aldehyde group of para-
o Reduction of inorganic S compounds through the
dimethylaminobenzaldehyde producing a deep red color
bacterial enzyme cysteine desulfurase with its
Indicator Contents
coenzyme pyridoxyl phosphate
Kovacs reagent isoamyl alcohol
p-dimethylaminobenzaldehyde
conc. HCl
Ehrlichs reagent 190mL 95% ethyl alcohol
2g p-dimethylaminobenzaldehyde
40mL conc. HCl
H2S is detected by incorporating a heavy metal (i.e. Fe)
into the medium, producing a black precipitate (iron
sulfide, FeS)

Klieglers Iron Agar

o excellent medium for detecting glucose and lactose Reduction of Nitrate
fermentation Oxygen in nitrate is used as a hydrogen acceptor during
o Iron salts anaerobic respiration through the enzyme nitratase
o Glucose o
o Lactose o Nitrate is reduced to nitrite
o Phenol red METHOD
Medium Indicator Result Inoculate nitrate broth tube. Incubate.
KIA Ferrous sulfate Black/brown surface Add the 2 reagents (sulfanilic acid & dimethyl-alpha-
Pb acetate agar Lead acetate Black/brown surface naphthylamine).
SIM medium o red: (+)
o Sulfur Reduction, Indole Production, Motility o NVR: add zinc powder
o Contains ferrous ammonium sulfate and sodium red: (-); organism was not able to reduce nitrate
thiosulfate, casein peptone, small amount of agar, to nitrite but the zinc did
and animal tissue (for amino acids and nutrients) NVR: (+); nitrate was reduced to ammonia or
nitrogen (beyond nitrite production)
Decomposition of Urea no satisfactory test for 2,3-butanediol formation
Urea Broth acetoin/acetyl-methyl-carbinol
o A buffered solution of yeast extract and urea o precursor of 2,3-butanediol
o Sterilized by filtration because it is heat-labile o detected by Barritts reagent
Urease causes urea to be broken down into ammonia and VP reagent A: alpha-napthol + VP reagent B: 40%
CO2 KOH
medium pink to red in the presence of acetoin
Enterobacter, Serratia, Erwinia, Bacillus, Aeromas
Shake, let stand 15 min (often necessary to re-incubate
for several hours)
o Ammonia increases the pH of the broth
Present in bacteria of genera Proteus, Providencia,
Morganella
Indicator Result
Phenol red (+): pinkish-red/cerise
Citrate Utilization
pH < 6.8: YELLOW
Some organisms (Salmonella typhimurium, E. aerogenes)
pH > 8.1: RED
utilize citrate as an organic carbon source
Formation of alkali end products resulting in a Prussian
blue color
Phenolphthalein (+): pink-red Nitrogen is obtained from ammonium salts
pH < 8.1: COLORLESS Medium Indicator Additional Notes
pH > 8.1: pink-red Simmons Bromothymol blue has NH4H2PO4
Methyl Red & Voges Proskauer Tests Citrate pH < 6.9 GREEN yellow: still (+) since
MRVP medium contains glucose which can be fermented pH > 7.6 - BLUE citrate was utilized but
by gram-negative intestinal bacteria products were not alkali
End products differentiate gram-negative bacteria Kosers TURBIDITY mainly used to
MR Test / Mixed Acid Fermentaion Test Citrate differentiate Escherichia
Bacteria ferment glucose, producing large amounts of and Enterobacter
lactic acid, acetic acid, succinic acid, formic acid, CO2, H2, Enterobacter: can utilize
and ethanol which can reduce the pH of the medium to 5 citrate, causing turbidity
or below
If the medium turns red after adding MR, the organism is
a mixed acid fermenter which can produce more gas due
to formic hydrogenylase
Present in Escherichia, Salmonella, Proteus, Aeromonas

Catalase Production
MR: pH < 4.4 red pH > 6.0 yellow Some bacteria produce catalase to survive in the
some (-) in MR may be due to increased production of presence of H2O2 which is toxic
butanediol instead of mixed acids thus will test positive
for VP test
Catalase-Positive:

VP Test / Butanediol Formation
bacteria ferment glucose, producing non-acid end-
(+) result: bubbles effervescing (rapid and sustained)
products, 2,3-butanediol and ethanol, which result in less
(-) if bubbles form after 20-30 seconds
lowering of the pH (negative MR test)

kap