BACTERIAL IDENTIFICATION BILE SOLUBILITY TEST

METHODS AND STRATEGIES Purpose This test differentiates Streptococcus

pneumoniae (positive soluble) from alpha-hemolytic
ACETAMIDE UTILIZATION streptococci (negative insoluble).
Purpose - Differentiate microorganisms based on the Principle- Bile or a solution of a bile salt (e.g., sodium
ability to use acetamide as the sole source of carbon. desoxycholate) rapidly lyses pneumococcal colonies.
Principle -Bacteria capable of growth on this medium Lysis depends on the presence of an intracellular
produce the enzyme acylamidase, which deaminates autolytic enzyme, amidase. Bile salts lower the
acetamide to release ammonia. The production of surface tension between the bacterial cell membrane
ammonia results in an alkaline pH, causing the and the medium, thus accelerating the organism s
medium to change color from green to royal blue. natural autolytic process.

ACETATE UTILIZATION
Purpose - Differentiate organisms based on ability to BUTYRATE DISK
use Purpose - This is a rapid test to detect the enzyme
acetate as the sole source of carbon. Generally used butyrate esterase, to aid identification of Moraxella
to differentiate Shigella sp. from Escherichia coli. (Branhamella) catarrhalis.
Principle -This test is used to differentiate an Principle - Organisms capable of producing butyrate
organism capable of using acetate as the sole source esterase hydrolyze bromochlorindolyl butyrate.
of carbon. Organisms capable of using sodium Hydrolysis of the substrate in the presence of butyrate
acetate grow on the medium, resulting in an alkaline esterase releases indoxyl, which in the presence of
pH, turning the indicator from green to blue. oxygen spontaneously forms indigo, a blue to blue-
Positive: Escherichia coli violet color.
Negative: Shigella sonnei
CAMP TEST
BACITRACIN SUSCEPTIBILITY Purpose - The Christie, Atkins, and Munch-Peterson
Purpose -This test is used for presumptive (CAMP) test is used to differentiate group B
identification streptococci (Streptococcus agalactiae positive) from
and differentiation of beta-hemolytic group other streptococcal species. Listeria monocytogenes
A streptococci (Streptococcus pyogenes also produces a positive CAMP reaction.
susceptible) from other beta-hemolytic streptococci.It Principle- Certain organisms (including group B
is also used to distinguish streptococci) produce a diffusible extracellular
staphylococci species (resistant) from micrococci hemolytic protein (CAMP factor) that acts
susceptible). synergistically with the betalysin of Staphylococcus
Principle -The antibiotic bacitracin inhibits the aureus to cause enhanced lysis of red blood cells.
synthesisof bacterial cell walls. A disk (TaxoA) The group B streptococci are streaked perpendicular
impregnatedwith a small amount of bacitracin to a streak of S. aureus on sheep blood agar. A
(0.04units) is placed on an agar plate, allowing positive reaction appears as an arrowhead zone of
theantibiotic to diffuse into the medium and inhibit the hemolysis adjacent to the place where the two streak
growth of susceptible organisms. After incubation,the lines come into proximity
inoculated plates are examined forzones of inhibition
surrounding the disks. CATALASE TEST
Purpose - This test differentiates catalase-positive
Bacitracin (A disk) Susceptibility. Any zone of micrococcal and staphylococcal species from
inhibition is positive (Streptococcus catalase-negative streptococcal species.
pyogenes); growth up to the disk is negative Principle - Aerobic and facultative anaerobic
(Streptococcus agalactiae). organisms produce two toxins during normal
metabolism, hydrogen peroxide (H2O2) and
BILE ESCULIN TEST superoxide radical (O2?). These bacteria have two
Purpose - This test is used for the presumptive enzymes that detoxify the products of normal
identification of enterococci and organisms in the metabolism. One of these enzymes, catalase, is
Streptococcus bovis group. The test differentiates capable of converting hydrogen peroxide to water and
enterococci and group D streptococci from non group oxygen. The presence of the enzyme in a bacterial
D viridans streptococci. isolate is evidenced when a small inoculum
Principle - Gram-positive bacteria other than some introduced into hydrogen peroxide (30% for the slide
streptococci and enterococci are inhibited by the bile test) causes rapid elaboration of oxygen bubbles. The
salts in this medium. Organisms capable of growth in lack of catalase is evident by a lack of or weak bubble
the presence of 4% bile and able tohydrolyze esculin production.
to esculetin. Esculetin reacts with Fe3+ and forms a
dark brown to black precipitate. CETRIMIDE AGAR
Purpose - This test is primarily used to isolate and hydrolyze) an amino acid to form an amine.
purify Pseudomonas aeruginosa from contaminated Decarboxylation, or hydrolysis, of the amino acid
specimens. results in an alkaline pH and a color change from
Principle - The test is used to determine the ability of orange to purple.
an organism to grow in the presence of cetrimide, a Positive:
toxic substance that inhibits the growth of many Lysine Klebsiella pneumoniae
bacteria by causing the release of nitrogen and Ornithine Enterobacter aerogenes
phosphorous, which slows or kills the organism. P. Arginine Enterobacter cloacae
aeruginosa is resistant to cetrimide. Base
(+) GROWTH (-) NO GROWTH Negative:
COLOR CHANGE NO COLOR CHANGE Lysine Enterobacter cloacae
Ornithine Klebsiella pneumoniae
CITRATE UTILIZATION Arginine Klebsiella pneumoniae
Purpose - The purpose of this test is to identify Base Klebsiella pneumoniae
organisms capable of using sodium citrate as the sole
carbon source and inorganic ammonium salts as the DNA HYDROLYSIS (DNASE TEST AGAR)
sole nitrogen source. The test is part of a series Purpose This test is used to differentiate organisms
referred to as IMViC (indole, methyl red, Voges- based on the production of deoxyribonuclease. It is
Proskauer, and citrate), which is used to differentiate used to distinguish Serratia sp. (positive) from
Enterobacteriaceae from other gram-negative rods. Enterobacter sp., Staphylococcus aureus (positive)
Principle - Bacteria that can grow on this medium from other species, and Moraxella catarrhalis
produce an enzyme, citrate-permease, capable of (positive) from Neisseria sp.
converting citrate to pyruvate. Pyruvate can then enter Principle The test is used to determine the ability of
the organisms metabolic cycle for the production of an organism to hydrolyze DNA. The medium is pale
energy. Bacteria capable of growth in this medium use green because of the DNA methyl green complex. If
the citrate and convert ammonium phosphate to the organism growing on the medium hydrolyses
ammonia and ammonium hydroxide, creating an DNA, the green color fades and the colony is
alkaline pH. The pH change turns the bromthymol surrounded by a colorless zone.
blue indicator from green to blue.
ESCULIN HYDROLYSIS
CITRATE UTILIZATION Purpose This test is used for the presumptive
Positive: Enterobacter aerogenes identification and differentiation of
(ATCC13048) growth, blue color Enterobacteriaceae.
Negative: Escherichia coli (ATCC25922) little Principle This test is used to determine whether an
to no growth, no color change organism is able to hydrolyze the glycoside
esculin. Esculin is hydrolyzed to esculetin,
COAGULASE TEST which reacts with Fe3+ and forms a dark brown to
Purpose The test is used to differentiate black precipitate.
Staphylococcus aureus (positive) from coagulase-
negative ??staphylococci (negative). FERMENTATION MEDIA
Principle S. aureus produces two forms of coagulase, Purpose - Fermentation media are used to
bound and free. Bound coagulase, or clumping factor, differentiate organisms based on their ability to
is bound to the bacterial cell wall and reacts directly ferment carbohydrates incorporated into the basal
with fibrinogen. This results in precipitation of medium. Andrade s formula is used to differentiate
fibrinogen on the staphylococcal cell, causing the cells enteric bacteria from coryneforms, and bromocresol
to clump when a bacterial suspension is mixed with purple is used to distinguish enterococci from
plasma. The presence of bound coagulase correlates streptococci.
with free coagulase, an extracellular protein enzyme Principle Carbohydrate fermentation is the process
that causes the formation of a clot when S. aureus microorganisms use to produce energy. Most
colonies are incubated with plasma. The clotting microorganisms convert glucose to pyruvate during
mechanism involves activation of a plasma glycolysis; however, some organisms use alternate
coagulase-reacting factor (CRF), which is a modified pathways.
or derived thrombin molecule, to form a coagulase- A fermentation medium consists of a basal medium
CRF complex. This complex in turn reacts with containing a single carbohydrate (glucose, lactose, or
fibrinogen to produce the fibrin clot. sucrose) for fermentation. However, the medium may
contain various color indicators, such as Andrade s
DECARBOXYLASE TESTS (MOELLER S METHOD) indicator, bromocresol, or others. In addition to a color
Purpose This test is used to differentiat decarboxylase indicator to detect the production of acid from
producing Enterobacteriaceae from other gram fermentation, a Durham tube is placed in each tube to
negative rods. capture gas produced by metabolism.
Principle This test measures the enzymatic ability
(decarboxylase) of an organism to decarboxylate (or FLAGELLA STAIN (WET MOUNT TECHNIQUE)
Purpose This technique is used to visualize the are capable of hydrolyzing tryptophan to
presence and arrangement of flagella for the pyruvate,ammonia, and indole. Kovac s reagent
presumptive identification of motile bacterial species. (dimethylamine-benzaldehyde and hydrochloride),
Principle Flagella are too thin to be visualized using a when added to the broth culture, reacts with the
bright field microscope with ordinary stains, such as indole, producing a red color. An alternative method
the Gram stain, or a simple stain. A wet mount uses Ehrlich s reagent. Ehrlichs reagent has the same
technique is used for staining bacterial flagella, and it chemicals as the Kovac preparation, but it also
is simple and useful when the number and contains absolute ethyl alcohol, making it flammable.
arrangement of flagella are critical to the identification Ehrlich s reagent is more sensitive for detecting small
of species of motile bacteria. The staining procedures amounts of indole
require the use of a mordant so that the stain adheres
in layers to the flagella, allowing visualization. INDOLE PRODUCTION
Quality Control
GELATIN HYDROLYSIS A. Kovac s Method
Purpose The production of gelatinases capable of Positive: Escherichia coli
hydrolyzing gelatin is used as a presumptive test for Negative: Klebsiella pneumoniae
the identification of various organisms, including B. Ehrlich s Method
Staphylococcus sp., Enterobacteriaceae, and some Positive: Haemophilus influenzae
gram-positive bacilli. Negative: Haemophilus parainfluenzae
Principle This test is used to determine the ability of C. Ehrlich s Method (Anaerobic)
an organism to produce extracellular proteolytic Positive: Porphyromonas asaccharolytica
enzymes (gelatinases) that liquefy gelatin, a Negative: Bacteroides fragilis
component of vertebrate connective tissue.
A, Positive; note liquefaction at top of tube. LEUCINE AMINOPEPTIDASE (LAP) TEST
B, Uninoculated tube. Purpose The LAP test is used for the presumptive
identification of catalase-negative gram-positive cocci.
GROWTH AT 42 C Principle
Purpose This test is used to differentiate a The LAP disk is a rapid test for the detection of the
pyocyanogenic pseudomonads from other enzyme leucine aminopeptidase.
Pseudomonas sp. Leucinebetanaphthylamide–impregnated disks serve
Principle The test is used to determine the ability of an as a substrate for the detection of leucine
organism to grow at 42 C. Several Pseudomonas aminopeptidase. After hydrolysis of the substrate by
species have been isolated in the clinical laboratory the enzyme, the resulting beta-naphthylamine
that are capable of growth at elevated temperatures. produces a red color upon addition of
Positive: Pseudomonas aeruginosa cinnamaldehyde reagent.
Negative: Pseudomonas fluorescens Positive: Enterococcus faecalis - red color
Negative: Aerococcus viridans -no color change
HIPPURATE HYDROLYSIS
Purpose Production of the enzyme hippuricase is LITMUS MILK MEDIUM
used for the presumptive identification of a variety of Purpose This test differentiates microorganisms
microorganisms. based on various metabolic reactions in litmus milk,
Principle The end products of hydrolysis of hippuric including fermentation, reduction, clot formation,
acid by hippuricase include glycine and benzoic digestion, and the formation of gas. Litmus milk is also
acid.Glycine is deaminated by the oxidizing agent used to grow lactic acid bacteria.
ninhydrin, which is reduced during the process.The Principle This test is used to determine an organism’s
end products of the ninhydrin oxidation react to form a ability to metabolize litmus milk. Fermentation of
purple-colored product. The test medium must contain lactose is demonstrated when the litmus turns pink as
only hippurate, because ninhydrin might react with a result of acid production. If sufficient acid is
any free amino acids present in growth media or other produced, casein in the milk is coagulated, solidifying
broths. the milk. With some organisms, the curd shrinks and
Positive: Deep purple color whey is formed at the surface. Some bacteria
Negative: Colorless or slightly yellow pink color hydrolyze casein, causing the milk to become straw
Positive: Streptococcus agalactiae colored and resemble turbid serum. Additionally, some
Negative: Streptococcus pyogenes organisms reduce litmus, in which case the medium
becomes colorless in the bottom of the tube
INDOLE PRODUCTION
Purpose This test is used to identify organisms that LITMUS MILK MEDIUM-QUALITY CONTROL
produce the enzyme tryptophanase. Fermentation: Clostridium perfringens – gas
Principle The test is used to determine an organisms production
ability to hydrolyze tryptophan to form the compound Acid: Lactobacillus acidophilus -clot formation
indole. Tryptophan is present in casein Peptonization: Pseudomonas aeruginosa – clearing
and animal protein. Bacteria with tryptophanase
LYSINE IRON AGAR (LIA) some members further metabolize nitrite to other
Purpose This test is used to differentiate gram- compounds.
negative bacilli based on decarboxylation or
deamination of lysine and the formation of hydrogen O-NITROPHENYL-?-D-GALACTOPYRANOSIDE
sulfide (H2S). (ONPG) TEST
Principle Lysine iron agar contains lysine, peptones, a Purpose This test is used to determine the ability of
small amount of glucose, ferric ammonium citrate, and an organism to produce ?-galactosidase, an enzyme
sodium thiosulfate. The medium has an aerobic slant that hydrolyzes the substrate ONPG to form a visible
and an anae bic butt. Whenglucose is fermented, the (yellow) product, orthonitrophenol. The test
butt of the medium becomes acidic (yellow). If the distinguishes late lactose fermenters from non–
organism produces lysine decarboxylase, cadaverine lactose fermenters of Enterobacteriaceae.
is formed. Cadaverine neutralizes the organic acids Positive: Yellow (presence of ?-galactosidase)
formed by glucose fermentation, and the butt of the Negative: Colorless (absence of enzyme)
medium reverts to the alkaline state (purple). If the
decarboxylase is not produced, the butt remains OPTOCHIN (P DISK) SUSCEPTIBILITY TEST
acidic (yellow). If oxidative deamination of lysine Purpose This test is used to determine the effect of
occurs, a compound is formed that, in the presence of Optochin (ethyl hydrocupreine hydrochloride) on an
ferric ammonium citrate and a coenzyme, flavin organism. Optochin lyses pneumococci (positive test),
mononucleotide, forms a burgundy color on the slant. but alpha-streptococci are resistant (negative test).
If deamination does not occur, the LIA slant remains Positive: Streptococcus pneumoniae
purple. Bromocresol purple, the pH indicator, is yellow Negative: Streptococcus pyogenes
at or below pH 5.2 and purple at or above pH 6.8.
OXIDASE TEST (KOVAC’S METHOD)
METHYL RED/VOGES-PROSKAUER (MRVP) Purpose This test determines the presence of
TESTS cytochrome oxidase activity in microorganisms for the
Purpose The combination test methyl red (MR) and identification of oxidase-negative Enterobacteriaceae,
Voges-Proskauer (VP) differentiates members of the differentiating them from other gram-negative bacilli.
Enterobacteriaceae family.
OXIDATION/FERMENTATION (OF) MEDIUM
MICRODASE TEST (MODIFIED OXIDASE) (CDC METHOD)
Purpose This test is used to differentiate gram- Purpose This test is used to differentiate
positive, catalase-positive cocci (micrococci from microorganisms based on the ability to oxidize or
staphylococci). ferment specific carbohydrates.
Positive: Development of blue to purple-blue color
Negative: No color change PHENYLALANINE DEAMINASE AGAR Purpose
This test is used to determine the ability of an
MOTILITY TESTING organism to oxidatively deaminate phenylalanine to
Purpose These tests are used to determine whether phenylpyruvic acid. The genera Morganella, Proteus,
an enteric organism is motile. An organism must have and Providencia can be differentiated from other
flagella to be motile. members of the Enterobacteriaceae family
Positive: Green color develops on slant after ferric
MRS BROTH chloride is added .
Purpose This test is used to determine whether an Negative: Slant remains original color after the
organism forms gas during glucose fermentation. addition of ferric chloride
Some Lactobacillus spp. and Leuconostoc sp.
produce gas. L-PYRROLIDONYL ARYLAMIDASE (PYR) TEST
Purpose This test is used for the presumptive
4-METHYLUMBELLIFERYL-?-D-GLUCURONIDE identification of group A streptococci (Streptococcus
(MUG) TEST pyogenes) and enterococci by the presence of the
Purpose This test is used to presumptively identify enzyme L-pyrrolidonyl arylamidase.
various genera of Enterobacteriaceae and verotoxin- Positive: Bright red color within 5 minutes
producing Escherichia coli. Negative: No color change or an orange color
Positive: Escherichia coli
Negative: Klebsiella pneumoniae PYRUVATE BROTH
Positive: Electric blue fluorescence Purpose This test is used to determine the ability of
Negative: Lack of fluorescence an organism to utilize pyruvate. This aids in the
differentiation between Enterococcus faecalis
NITRATE REDUCTION (positive) and Enterococcus faecium (negative).
Purpose This test is used to determine the ability of
an organism to reduce nitrate to nitrite. All members of SALT TOLERANCE TEST
the Enterobacteriaceae family reduce nitrate, but Purpose This test is used to determine the ability of
an organism to grow in high concentrations of salt. It
is used to differentiate enterococci (positive) from In broth media, nutrients are dissolved in water, and
nonenterococci (negative). Positive: Visible turbidity bacterial growth is indicated by a change in the
in the broth, with or without a color change from broth’s appearance from clear to turbid (i.e., cloudy).
purple to yellow More growth indicates a higher cell density and
Negative: No turbidity and no color change greater turbidity.
At least 106 bacteria per mL of broth are needed for
SPOT INDOLE TEST turbidity to be detected with the unaided eye.
Purpose This test is used to determine the presence
of the enzyme tryptophanase. It is a rapid method that Classification of Media
can be used in lieu of the tube test. Based on the consistency:
Positive: Development of a blue color Solid media
Within 20 seconds Semisolid media
Liquid Media
TRIPLE SUGAR IRON AGAR (TSI) Based on the function or property:
Purpose TSI is used to determine whether a gram Simple media
negative rod ferments glucose and lactose or sucrose Enriched media
and forms hydrogen sulfide (H2S). The test is used Enrichment broth
primarily to differentiate members of the Selective media
Enterobacteriaceae family from other gram-negative Indicator media
rods. Differential media
Composite media
UREASE TEST (CHRISTENSEN’S METHOD) Transport media
Purpose This test is used to determine an organism’s
ability to produce the enzyme urease, which Agar or agar – agar – Transport media
hydrolyzes urea. Proteus sp. may be presumptively Peptone - mixture of partially digested proteins
identified by the ability to rapidly hydrolyze urea. Yeast or meat extract
Positive: Change in color of slant from light orange to NaCl
magenta. Melting point : 98°C
Negative: No color change (agar slant and butt Solidifying point : 42°C
remain light orange) % of agar : Solid media (2%)

X AND V FACTOR TEST Simple Media

Purpose The X and V factor test is used to Also called Basal medium
differentiation Most commonly used in routine labs.
Haemophilus species. Members of the genus e.g. Nutrient broth, NA
Haemophilus require accessory growth factors in
vitro. Some Haemophilus spp. require X factor Enriched Media
(hemin) alone, V factor (nicotinamide adenine Blood, serum or egg added to the basal medium.
dinucleotide [NAD]) alone, or a combination of the To grow bacteria which are more exacting in
two. nutritional requirements.
e.g. Blood Agar, Chocolate Agar
Culture Media
Indications/ Need for culture For mixed cultures or materials containing more than
 Isolate bacteria in pure cultures. one bacterium. Contains substances which
 Demonstrate their properties. stimulates wanted bacteria & inhibits unwanted
 Obtain sufficient growth for preparation of bacteria.
antigens & for other tests. e.g. Tetrathionate broth
 Typing bacterial isolates.
 Antibiotic sensitivity. Selective Media
 Estimate viable counts. Substances added to a solid media to favour the
 Maintain stock cultures. growth of wanted bacteria. e.g. MacConkey agar
media
To grow and isolate all bacteria present in a clinical
specimen Indicator Media
• To determine which of the bacteria that grow are Change in the colour of indicator with the growth of
most likely causing infection and which are likely bacteria.
contaminants or colonizers e.g. Sulphite in Wilson Blair media for
• To obtain sufficient growth of clinically relevant Salmonella typhi.
bacteria to allow identification, characterization, and
susceptibility testing Differential Media
To bring out differing characteristics of bacteria.
e.g. MacConkey’s agar
 media. Some carbohydrate and mineral salts are
Differential –Identify hemolysis -Some bacteria usually added as well.
secrete enzymes that lyse red blood cells
(hemolysins) such that a clearing around the colony A. According to Composition:
appears.  Chemically-defined media are those in
Beta hemolysis-complete clearing (white hemolysis) which the exact chemical composition are
Alpha hemolysis –incomplete clearing (green known. For the growth of autotrophic
hemolysis) microbes and microbial assays. Table 1-1
Gamma hemolysis-no hemolysis shows an example of a chemically-defined
medium.
Table 1-1 chemically-defined medium for growing a
typical chemoheterotrophs, such as Escherichia coli.
Composite Media Constituent Amount
Bacteriological culture media indicating a variety of Glucose 5.0g
growth effects (such as carbohydrate fermentation, Ammonium Phosphate, monobasic 1.0g
indole production, etc.) Sodium Chloride 5.0g
For identification of isolates Magnesium Sulfate
e.g. Triple Sugar Iron 0.2g
Potassium Phosphate 1.0g
Transport Media Water 1 L____
For delicate organisms
Delay in transit  Complex Media are natural media like Milk,
e.g. Stuart’s transport media blood and other nutrients derived from
animal, plant and yeast extracts (table 1-2).
Exact compositions vary depending on the
QUANTITATIVE STREAKING PATTERN source.

INCUBATION CONDITIONS Table 1-2 a complex medium for the growth of

Aerobes grow in ambient air which contains 21% O2 heterotrophic bacteria
and 0.03% CO2.
Anaerobes in an environment composed of 5-10% Constituent Amount
H2, 5-10% CO2, and 0% O2.
Capnophiles like H. influenzae & N. gonorrhea Peptone 5.0g
require 5-10% CO2 and about 15% O2. Beef extract 3.0g
Microaerophiles grow at reduced (5-10%) O2 and Sodium Chloride 8.0g
increased (8-10%) CO2 as well as at higher Agar 15.0g
temperature (42 C), Water 1
L_____
ISOLATION OF BACTERIA
COLONY CHARACTERISTICS B. According to Purpose
Colony size (pinpoint, small, medium, large) *General-purpose media will support a large number
Colony pigmentation of organisms.
Colony shape (form, elevation and margin) *Selective media provide nutrients that enhance the
Colony surface appearance (glistening, opaque, dull, growth and predominance of a particular type of
transparent) microbe while unwanted types are
Changes in agar media resulting from bacterial growth suppressed.
(hemolysis on bap, changes in color of pH indicator, *Differential media allow distinctive growth of various
pitting on agar surface wanted microbes making them easy to identify.
Odor *Reducing media support growth of obligate
anaerobes. Sodium thioglycollate depletes the
Growing microorganisms in vitro requires mixtures of medium of oxygen by combining with
nutrients known as culture media. Media may serve dissolved oxygen in the medium.
many different purposes in microbial work. Media are *enrichment media are media designed to increase
classified based on their composition, physical state, numbers of wanted microbes in a detectable level.
and purpose. Most media designed for the initial This is used to grow fastidious
growth and isolation of microorganisms are rich in microbes.
protein components derived from animal meats. Many
bacteria are unable to break down proteins to usable C. According to State:
forms and must be provided with extracted or partially *Solid media have agar-agar or gelatin added to them
degraded protein materials (peptides, proteoses, as solidifying agent. They are used for the study of
peptones, amino acids). Meat extracts, or partially colony morphology, isolation of
cooked meats, are the basic nutrients of many culture pure cultures and prolong storage of pure cultures.
*Semisolid media have lesser amount of agar, thus electric stove to accomplish this. You must stir the
they have a jelly-like consistency. These are used in media almost constantly with a glass stirring rod to
sugar utilization and motility tests. prevent burning or boiling over.
*broths are liquid media lacking solidifying agents.
Table 1-3 summary of the amounts of medium to be
Preparation of Media used in various culture preparations.
Essential to the preparation of microbiological media
is the accurate weighing of powders and granular Medium Amounts Autoclaving
substances. When dehydrated media are container
reconstituted, a specific number of grams of the Broth 5 – 10 Screw-capped
powdered medium is added to distilled water and mL Culture tube
heated to dissolve the powder completely. If you have
Agar 10 mL Screw-capped
not weighed the powder and measured accurately,
slants Culture tube
you will not have the proper proportion of nutrients in
Agar 12 mL Screw-capped
the finished medium. If you do not weigh solid media
deeps Culture tube
accurately, you run the risk of having the medium turn
Agar 7 mL Screw-capped
out semisolid or too dry.
Stabs Culture tube
A triple beam balance is generally used since large
amounts are involved. When you weigh powders or Agar 200 mL 500 mL
granular materials, you must never put them directly plates (20-25 Erlenmeyer’s
on the weighing platform of the balance. You need to mL/petri flask
tightly close the bottle for dehydrated powder are dish) (2/3 full)
highly hygroscopic. It will absorb moisture from the air
and will cause the hardening of the media.
For amounts up to 15g, you should use glazed Agar that is to be used for culture plates is
weighing paper to hold the powder media you are dispensed into 500 mL flask. This agar in flask, after
weighing, and for amounts larger than this you should autoclaving, can be poured into several Petri dishes to
use small beaker or weigh boat. make culture plates. A culture plate contains
The directions on the labels of most commercially approximately 20 to 25 mL of sterile agar medium.
prepared media bottles give you the amount of Media are never sterilized in the autoclave while in
powdered medium to be re-hydrated in 1000mL (1L) petri dish because the agar would bubble out of the
of distilled water. The amount of powdered medium plate (petri dish).
per liter of water varies for each medium. Therefore,
be sure to read the label for each type of medium. You Form the habit of rinsing with hot water all
rarely need to make 1000 mL of medium, so you need containers that have held agar as soon as they are
to calculate for smaller amounts. emptied. This is especially important for dispensing
There are other methods in solving for the exact pipettes because narrow openings can become
amount of powder but in this exercise a direct clogged as the agar solidifies.
proportion will be used. In this method you must set
up a ratio between the grams/liter directions on the When media are sterilized in a steam
label and the amount of medium you wish to make. sterilizer such as autoclave, they are superheated
For example, if the label on the bottle directs you to under pressure; that is the boiling point is raised as
add 15g of medium to 1000 mL of distilled water, how the steam pressure increases in the sealed sterilizer.
many grams of powdered medium should you weigh Under these conditions, a tightly stoppered container
to make 200 mL? could explode when the medium expands because of
Solve for X: the extreme heat. You must always leave about 2
___15g____ = Xg___ inches of air space between the agar level and the
1000mL 200mL neck of the flask for this same reason. Even with the
capped loose, the expanding agar can boil over top of
1000X = (15)(200) the bottle if you do not leave enough air space. If you
are using cotton-stoppered flasks, the problem is
3000 eliminated because the air escapes freely.
X = -------------
1000

X = 3g

The graduated cylinder is the basic

measuring device for liquids. Use appropriate size for
the right amount of distilled water. Agar media must
be heated to boiling for several minutes before they
will dissolve completely. Use a double boiler and the