Jescika Vidal MicroLab Final

4/17/2011
Selective Media for Isolation of Gram Positive Cocci

Gram negative organisms frequently inhibit growth of Gram positives when they are cultivated together. Therefore,
when looking for staphylococci or streptococci in a clinical sample, it may be necessary to begin by streaking the
unknown mixture onto a selective medium that inhibits Gram negative growth. The selective media introduced in this
unit to isolate (and sometimes presumptively identify) streptococci and staphylococci n human samples. Phenylethyl
Alcohol Agar PEA and Mannitol Salt agar MSA is a selective and differential medium developed to favor growth of
staphylococcus and to differentiate pathogenic from nonpathogenic members of the genus.

Mannitol Salt Agar MSA

Mannitol Salt Agar MSA contains the carbohydrate mannitol, 7.5% NaCl, and the pH indicator phenol red. Phenol red is
yellow below pH 6.8, red at pH 7.4 to 8.4, and pink at pH 8.4 and above, The NaCl makes the medium selective for
staphylococci because most other bacteria cannot survive in this level of salinity. The pathogenic species of
Staphylococcus ferment mannitol and produce acid, which turns the pH indicator yellow. Nonpathogenic staphylococcal
species grow but produce no color change. The development of yellow halos around the bacterial growth provides
strong evidence that the organism is pathogenic usually staphylococcus aureus. Good growth that produce no color
change is evidence for nonpathogenic Staphylococcus. With few exceptions, organisms that grow poorly on the medium
are not staphylocci. MSA is used for isolation and differentiation of pathogenic Staphylococcus species, principally S.
aureus.

Selective Media for Isolation of Gram-Negative Rods

Hektoen Enteric Agar Hekton

HE Agar is an undefined medium designed to isolate Salmonella and Shigella species from other enteric based on the
ability to ferment lactose, sucrose, or salicin, and to reduce sulfur to hydrogen sulfide gas (H2S). In addition to the 3
sugars, sodium thioosulfate is included as a source of sulfur. Ferric ammonium citrate is added to react with H2S and
form black precipitate. Bile salts are included to inhibit most Gram positive cocci. Brromthymol Blue and acid fuchsin
dyes are added as color indicators. Differentiation is possible as a result of the various colors produced in the colonies
and in the agar. Enterics that produce acid from fermentation will produce yellow to salmon pink colonies. Organisms
such as Salmonella, Shigella, and Proteus that do not ferment any of the sugars produce blue green colonies. Proteus
and Salmonella species that reduce sulfur to H2S form colonies containing a black precipitate. HE agar is used to isolate
and differentiate Salmonella and Shigella species from other Gram negative enteric organisms.

MacConkey Agar MAC

MAC agar is a selective and differential medium containing lactose, bile salts, neutral red, and crystal violet. Bile salts
and crystal violet inhibit growth of Gram positive bacteria. Neutral red dye is a pH indicator that is colorless about a pH
of 6.8 and red at a pH less than 6.8. Acid accumulating from lactose fermentation turns the dye red. Lactose fermenters
turn a shade of red on MAC agar, whereas lactose nonfermenters retain their normal color or the color of the medium.
Formulations without crystal violet allow growth of Enterococcus and some species of Staphylococcus, which ferment
the lactose and appear pink on the medium, MAC agar is used to isolate and differentiate members of the
Enterobacteriaceae based on the ability to ferment lactose. Variations on the standard medium include MAC Agar
without Crystal Violet to allow growth of Gram positive cocci, or MAC agar to CS to control swarming bacteria (Proteus)
to interfere with other results.

Fermentation Tests
Methyl Red and Voges- Proskaur Tests MR-VP
MRVP Broth is a combination medium used for both methyl red and voges proskaur test. It is a simple solution
containing only peptone, glucose, and a phosphate buffer. The peptone and glucose provide protein and fermentable
carbohydrate, respectively and the potassium phosphate resists pH changes in the medium. The MR test is designed to
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detect organisms capable of performing a mixed acid fermentation, which overcomes the phosphate buffer in the
medium and lowers the pH. The acids produced by these organisms tend to be stable, whereas acids produced by other
organisms tend to be unstable and subsequently are converted to more neutral products. Mixed acid fermentation is
verified by the addition of methyl red indicator dye following incubation. Methyl red is red at pH 4.4 and yellow at pH
6.2. Between these two pH values it is various shades of orange. Red color is the only true indication of a positive result;
orange is negative or inconclusive. The Voges Proskauer test was designed for ortanisms that are able to ferment
glucose, but quickly conver their acid products to acetoin and 2,3 butanediol. Addition of VP reagents to the medium
oxidizes the acetoin to diacetyl, which in turn reacts with guanidine nuclei from peptone to produce a red color. A
positive VP result, therefore is red. No color change or development of copper color is a result of interactions between
the reagents and should not be confused with the true red color of a positive result. Use of positive and negative
controls for comparison is usually recommended. After incubation, two 1 mL volumes are transferred from the MR-VP
broth to separate test tubes. Methyl red indicator reagent is added to one tube, and VP reagents are added to the other.
Color changes then are observed and documented. The methyl red and voges proskauer tests are components of the
IMViC battery tests (Indole, Methyl red, Voges Proskauer, and Citrate) used to distinguish members of the family
Enterobacteriacea and differentiate them from other gram negative rods.
Methyl Red

Citrate-Green Cap Slants

In many bacteria, citrate (citric acid) produced as acetyl coenzyme A (from the oxidation of pyruvate or the β-oxidation if
fatty acids) reacts with oxaloacetate at the entry to the Krebs cycle. Citrate then is converted back to oxaloacetate
through a complex series of reactions, which begins the cycle anew. In a medium in which citrate is the only available
carbon source, bacteria that possess citrate-permease can transport the molecules into the cell and produce pyruvate
(pyruvic acid) by a reversal of the above described reaction. Pyruvate then can be converted to a variety of products
depending on the pH of the environment. Simmons Citrate Agar is a defined medium that contains sodium citrate as the
sole carbon source and ammonium phosphate as the sole nitrogen source. Bromthyol blue dye, which is green at pH 6.9
and blue at pH 7.6, is added as an indicator. Bacteria that survive in the medium and utilize the citrate also convert the
ammonium phosphate to ammonia (NH3) and ammonium hydroxide (NH4OH), both of which tend to alkalinize the agar.
As the pH goes up, the medium changes from green to blue. Thus, conversion of the medium to blue is a positive citrate
test result. Occasionally a citrate positive organism will grow on a Simmons Citrate slant without producing a change in
color. In most cases, this is because of incomplete incubation. In the absence of color change, growth on the slant
indicates that citrate is being utilized and is evidence of a positive reaction. To avoid confusion between actual growth
and heavy inoculums, which may appear as growth, citrate slants typically are inoculated lightly with an inoculating
needle rather than a loop. The citrate utilization test is used to determine the ability of an organism to use citrate as its
sole source of carbon. Citrate utilization is one part of a test series referred to as the IMViC that is used to distinguish
between members of the family Enterobacteriaceae and differentiate them from other Gram negative rods.

Tests Detecting the presence of hydrolytic enzymes

Reactions that use water to split complex molecules are called hydrolysis reactions. The enzymes required for these
reactions are called hydrolytic enzymes. When the enzyme catalyzes its reaction inside the cell, intracellular. Enzymes
secreted from the cell to catalyze reactions in the external environment are called extracellular enzymes, or
exoenzymes. In this unit you will be performing tests that identify the actions of both intracellular and extracellular
types of enzymes. The hydrolytic enzymes detected by the Urease and Bile Esculin Tests are intracellular and produce
identifiable color changes in the medium. Nutrient Gelatin detects gelatinase- an exoenzyme that liquefies the solid
medium. DNase Agar, Milk Agar, Tributyrin Agar, and Starch Agar are plated media that identify extracellular enzymes
capable of diffusing into the medium and producing a distinguishable halo of clearing around the bacterial growth.

BEA Bile Esculin Test

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Bile Esculine contains esculin, peptone, bile, sodium azide, and ferric citrate. Esculin is a glycoside composed of glucose
and esculetin. Peptone and esculin provide nutrients, and bile inhibits growth of esculin provide nutrients, and bile
inhibits the growth of gram positive organisms. Ferric citrate is included as an indicator. Esculin can be easily hydrolyzed
under acidic conditions, but few bacteria outside of Group D streptococci and enterococci are able to do so in the
presence of bile. When the esculin molecules are split, esuletin reacts with ferric citrate and forms a dark brown
phenolic iron complex. Group D streptococci and enterococci grow well on the medium and usually darken it in 18 to 24
hours. Therefore, an organism that has not darkened more than half the medium after 48 hours considered negative. A
slant that is more than half darkened at any time within 48 hours can be recorded as completed positive. This test is
most commonly used for presumptive identification of enterococci and members of the Streptococcus bovis group, all
which are positive.

Urease
Urea is the product of decarboxylation of certain amino acids. It can be hydrolyzed to ammonia and carbon dioxide by
bacteria containing the enzyme urease. Many enteric bacteria (and few others) possess the ability to metabolize urea,
but only members of Proteus, Morganella, and Providencia are considered rapid urease positive organisms. Urea Agar
was formulated to differentiate rapid urease positive bacteria from slower urease positive bacteria. It contains urea,
peptone, potassium phosphate, glucose, and phenol red. Peptone and glucose provide essential nutrients for a broad
range of bacteria. Potassium phosphate is a mild buffer used to resist alkalinization of the medium from peptone
metabolism. Phenol red, which is yellow or orange below pH 8.4 and red or pink above, is included as an indicator. Urea
hydrolysis to ammonia by urease positive organisms will overcome the buffer in the medium and change it from orange
to pink within 24 hours. Weak positives may take several days. Urease negative organisms either produce no color
change in the medium or turn it yellow from acid products. Urea broth differs from urea agar in two important ways.
First its only nutrient source is a trace of yeast extract. Second it contains buffers strong enough to inhibit alkalinization
of the medium by all but the rapid urease positive organisms mentioned above. Other organisms even some that
ordinarily would be able to metabolize urea cannot survive the severe nutrient limitations or overcome the buffers in
urea broth. Pink color in the medium in less than 24 hours indicates a rapid urease positive organism. Orange or yellow
is negative. This test is used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease.
Urinary tract pathogens from the genus Proteus may be distinguished from other enteric bacteria by their rapid urease
activity.

DNAse Test
An enzyme that catalyzes the depolymerization of DNA into small fragments is called a deoxyribonuclease or DNase.
Ability to produce this enzyme can be determined by culturing and observing an organism on a DNase Test Agar Plate.
DNase Test Agar contains and emulsion of DNA, peptides as a nutrient source, and methyl green dye. The dye and
polymerized DNA form a complex that gives the agar a blue green color. Bacterial colonies that secrete DNase will
hydrolyze DNA in the medium into smaller fragments unbound from the methyl green dye. This results in clearing
around the growth. DNase Test Agar is used to distinguish Serratia species from Entero bacter species, Moraxella
catarrhalis from Neisseria species, and Staphylococcus aureus from other Staphylococcus species.

Blood
Several species of Gram positive cocci produce exotoxins called hemolysins, which are able to destroy red blood cells
and hemoglobin. Blood Agar which is a mixture of Tryptic Soy Agar and sheep blood, allows differentiation of bacteria
based on their ability to hemolyze RBCs. The three major types of hemolysis are β hemolysis, α hemolysis, and γ
hemolysis. β hemolysis is the complete destruction of RBCs and hemoglobin, and results in a clearing of the medium
around the colonies. α hemolysis is the partial destruction of RBS and produces a greenish discoloration of the agar
around the colonies. γ hemolysis is actually non-hemolysis and appears as simple growth with no change to the
medium. Hemolysins produced by streptococci are called streptolysins. They come in two forms- type O and type S.
Streptolysin O is oxygen labile and expresses maximal activity under anaerobic conditions. Streptolysin S is oxygen stable
but expresses itself optimally under anaerobic conditions as well .The easiest method of providing an environment
favorable for streptolysins on Blood Agar is what is called the streak stab technique. In this procedure the Blood Agar is
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streaked for isolation and then stabbed with a loop. The stabs encourage streptolysin activity because of the reduced
oxygen concentration of the subsurface environment. Blood agar is used for isolation and cultivation of many types of
fastidious bacteria. It also is used to differentiate bacteria based on their hemolytic characteristics, especially within the
genera Streptococcus, Enterococcus, and Aerococcus.

Coagulase Tests Rabbit Plasma

Staphylococcus aureus is an opportunistic pathogen that can be highly resistant to both the normal immune response
and antimicrobial agents. Its resistance results, in part, from the production of a Coagulase enzyme. Coagulase works in
conjunction with normal plasma components to form protective fibrin barriers around individual bacterial cells or groups
of cells shielding them from phagocystosis and other types of attack. Coagulase enzymes occur in two forms- bound
coagulase and free coagulase. Bound coagulase, also called clumping factor is attached to the bacterial cell wall and
reacts directly with fibrinogen in a plasma. The fibrinogen then precipitates, causing the cells to clump together in a
visible mass. Free coagulase is an extracellular enzyme (released from the cell) that reacts with a plasma component
called coagulase reacting factor CRF. The resulting reaction is similar to the conversion of prothrombin and fibrinogen in
the normal clotting mechanism. Two forms of the Coagulase Test have been devised to detect the enzymes: the Tube
Test and the Slide Test. The Tube test detects the presence of either bound or free coagulase, and the Slide Test detects
only bound coagulase. Both tests utilize rabbit plasma treated with anticoagulant to interrupt normal clotting
mechanisms. The Tube Test performed by adding the test organism to rabbit plasma in a test tube. Coagulation of the
plasma (including any thickening or formation of fibrin threads) within 24 hours indicates a positive reaction. The plasma
typically is examined for clotting (without shaking) periodically for about 4 hours. After 4 hours, coagulase negative
tubes can be incubated overnight but no more than a total of 24 hours because coagulation can take place early and
revert to liquid within 24 hours. In the Slide Test, bacteria are transferred to a slide containing a small amount of plasma.
Agglutination of the cells on the slide within 1 to 2 minutes indicates the presence of bound coagulase. Equivocal or
negative slide test results typically are given the test tube for confirmation. The Coagulase Test typically is used to
differentiate Staphylococcus aureus from other gram positive cocci.

Motility
Motility Test Medium is a semisolid medium designed to detect bacterial motility. Its agar concentration is reduced from
the typical 1.5% to 0.4%- just enough to maintain its form while allowing movement of motile bacteria. It is inoculated
by stabbing with a straight transfer needle. Motility is detectable as diffuse growth radiating from the central stab line. A
tetrazolium salt TTC is sometimes added to the medium to make interpretation easier. TTC is used by the bacteria as an
electron acceptor. In its oxidized form, TTC is colorless and soluble; when reduced it is red and insoluble. A positive
result for motility is indicated when the red reduce TTC is seen radiating outward from the central stab. A negative result
shows red only along the stab line. This test is used to detect bacterial motility. Motility is an important differential
characteristic of Enterobacteriaceae.

Tests Identifying Microbial Ability to Respire

In this unit we will examine several techniques designed to differentiate bacteria based on their ability to respire. As
mentioned earlier in this section, respiration is the conversion of glucose to energy in the form of ATP by way of
glycolysis, the Krebs cycle, and oxidative phosphorylation, in the electron transport chain (ETC). In respiration, the final
electron acceptor at the end of the ETC is always an inorganic molecule. Tests that identify an organism as an aerobic or
an anaerobic respirer generally are designed to detect Pacific products of (or constituent enzymes used in) the reduction
of the final electron acceptor (FEA). Aerobic respirers reduced oxygen to water or other compounds. Anaerobic respirers
reduce other inorganic molecules such as nitrate or sulfate. Nitrate is reduced to nitrogen gas (N2) or other nitrogenous
compounds such as nitrite (NO2), and sulfate is reduced to hydrogen sulfide gas (H2S). The exercises and organisms
chosen for this section directly or indirectly demonstrate the presence of an ETC. The catalase test detects organismal
ability to produce catalase – an enzyme that detoxifies the cell by converting hydrogen peroxide produced in the ETC to
water and molecular oxygen. The oxidase test identifies the presence of cytochrome C oxidase in the ETC.

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Catalse Test

The electron transport chains of aerobic and facultatively anaerobic bacteria are composed of molecules capable of
accepting and donating electrons as conditions dictate. As such, these molecules alternate between the oxidized and
reduced form, passing electrons down the chain to the final electron acceptor (FEA). Energy lost by electrons in this
sequential transfer is used to perform oxidative phosphorylation (I. E., Produce ATP from ADP). One carrier molecule in
the ETC called flavoprotein can bypass the next carrier in the chain and transfer of electrons directly to oxygen. This
alternate pathway produces two highly potent cellular toxins – hydrogen peroxide(H2O2) and super oxide radical(O2 -).
Organisms that produce these toxins also produce enzymes capable of breaking them down. Superoxide dismutase
catalyzes conversion of super oxide radicals (the more lethal of the two compounds) to hydrogen peroxide. Catalase
converts hydrogen peroxide into water and gaseous oxygen. Bacteria that produce catalase can be easily detected using
typical store grade hydrogen peroxide. When hydrogen peroxide is added to a catalase positive culture, oxygen gas
bubbles form immediately. If no bubbles appear, the organism is catalase negative. This test can be performed on a
microscope slide or by adding hydrogen peroxide directly to the bacterial growth. This test is used to identify organisms
that produce the enzyme catalase. It is used most commonly to differentiate members of the catalase positive
Micrococcaceae from the catalase negative Streptococcaceae. Variations on this test also may be used in identification
of mycobacterium species.

Oxidase Test

Bacterial respiratory electron transport chains capture energy in the form of high energy bonds and ATP via the
oxidation of NADH and FADH2 produced in the Krebs cycle and Glycolysis. The electron transport chains of many
aerobes, facultative anaerobes, and microaerophiles contain a carrier molecule called cytochrome C oxidase.
Cytochrome C oxidase is responsible for the transfer of electrons to oxygen, thus reducing it to water. The oxidase test is
performed using a chromogenic a reducing agent called tetramethyl- P-phenylenediamine to detect bacteria that
produce cytochrome C oxidase. Chromogenic reducing agents are chemicals that change or produce color as they
become oxidized. In the oxidase test, the chromogenic reducing reagent is added directly to bacterial growth on solid
media, or (more conveniently) a colony is transferred to paper saturated with reagent. Within seconds the reagents,
which acts as an artificial electron donor, will change color if oxidized cytochrome C is present. No color change within
the allotted time means no cytochrome oxidase is present and is a negative result. This test is used to identify bacteria
containing the respiratory enzyme cytochrome C oxidase. Among its many uses is the presumptive identification of the
oxidase positive Neisseria. It also can be useful in differentiating the oxidase negative Enterobacteriaceae from the
oxidase positive Pseudomonadaceae.

Antimicrobial Susceptibility and Resistance

Bacitracin, Novobiocin, Optochin Susceptibility Test

Antibiotics are antimicrobial substances produced by microorganisms. Bacitracin, produced by Bacillus licheniformis, is a
powerful peptide antibiotic that inhibits bacterial cell wall synthesis. Thus, it is effective only on bacteria that have cell
walls and are in the process of growing. The Bacitracin test is a simple test performed by placing a Bacitracin
impregnated disk on an agar plate inoculated to produce a bacterial lawn. The Bacitracin diffuses into the agar and,
where it's concentration is sufficient, inhibits growth of susceptible bacteria. Inhibition of bacterial growth will appear as
a clearing on the agar plate. Any zone of clearing 10 MM or greater around the disc is interpreted as Bacitracin
susceptibility. This test is used to differentiate and presumptively identify β-hemolytic group A streptococci
(streptococcus pyogenes) from other β-hemolytic group streptococci. It also differentiates the genus Staphylococcus
(resistant) from the susceptible Micrococcus and Stomatococcus. Novobiocin is an antibiotic, produced by Streptomyces
niveus. It interferes with ATPase activity and ultimately the production of ATP. Optochin is an antibiotic that interferes
with ATPase activity and ATP production in susceptible bacteria. The novobiocin test is used to differentiate coagulase
negative staphylococci. Most frequently used to presumptively identify the novobiocin resistant Staphylococcus
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saprophyticus. The optochin test is used to presumptively differentiate Streptoccous pneumoniae from other α-
hemolytic streptococci.

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Gram positive:
Staph epi (nonpathogen) (non lactose fermentor) (non hemo?) (cocci cluster)
Staph aureus (opportunistic) (pathogen) (mannitol fermentor) (β hemolysis) (cocci cluster)
Streps (chains)
Enterococci (strep) (chains)

Gram negative:
Enterobacteria

Lactose + (fermentor)
E. Coli (pink)
Klebsiella pneum (bad)
Serratia Marceans- color pigmented

Lactose – (nonfermentors)(disease)
Pseudomas aerginosa (clear)
Salmonella typh (H2S)
Shigella
Proteus (H2S)

Gra Catalas Oxidas Coagulas Hemolysi Lancefiel Optochi Bacitraci H2

  Bacteria m Shape e e e s d Group n n S I M V C

  E. coli - rod   -             + + - -
Lac Klebsiella - rod   -             - - + +
  Enterobacter - rod + -             - - + +
  Proteus - rod + -           + - + - +
Non
Lac Salmonella - rod   -           +        
  Pseduomans - rod   +                    
  Staphs aur + cocci +   + β                
  Staph epi + cocci +   - ϒ, non                
  Enterococcus + cocci -     α, non D N/A            
  Strep pneum + cocci -     α None Susc Resis          
Viridans
  Group + cocci -     α, non None Resis            
  Strep Agal + cocci -     β B N/A            
  Strep pyogen + cocci -     β A N/A            

Streps- grouped on hemolysis, biochemical, and serological . 3 groups based on hemolysis produced
α- s. Pneum, Viridans
β-S pyogenes & S agalactiae
ϒ- Enterococci
Biochemical
Sensitivity to Bacitratin- Group A (s Pyogenes) from other B
Sensitivity to Optochin- S. Pneum vs. Viridans

Serology- antigenic differences in cell wall carbs

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Group A- S pyogens
Group B- S agalactiae
Group D- enterococci
Viridans- S mutans, S mitis
S pneumo- no group