Lab Exercise: Staphylococcal, Streptococcal & Enteric...

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Lab Exercise: Staphylococcal, Streptococcal & Enteric Unknowns

OBJECTIVES 1. Identify each of the organisms worked with in this lab exercise. 2. Understand the use of differential and selective media in the identification of bacteria and the enrichment of such cultures for organisms of interest. 3. Understand the inoculations used throughout this laboratory exercise, use Table 4 as a study tool. 4. Understand the use of a dichotomous key and other data (including data tables) in determining species identification. BACKGROUND Staphylococcus The identification of bacterial pathogens, particularly from closely related non-pathogens is extremely important to the field of medical microbiology. In order to do this, selective and differential media are often used. Particularly when members of the normal flora can be opportunistic pathogens, it is important to have on hand easy ways of identification so that useful and appropriate treatment can be begun. The bacterial genus Staphylococcus contains three species of medical importance: S. aureus, S. epidermidis and S. saprophyticus. Of these three, only S. aureus is typically considered to be pathogenic, except under special circumstances. All three, however, can be found residing on the skin and mucous membranes of healthy adults. Staphylococci are particularly well suited to this environment because they are facultative halophiles and can grow in media containing up to 10% NaCl. This capability can be used to isolate staphylococci from a mixed culture of organisms or from a patient sample It is important to note, however, that microscopic examination must confirm the presence of non-spore forming staphylococci before biochemical testing can be performed. Once a staphylococcal organism has been isolated, the species can be determined by performing a few simple biochemical tests, including mannitol fermentation. Because both salt tolerance and mannitol fermentation are useful criteria for identifying members of the staphylococci, a medium which examines both of these characteristics is commonly used, Mannitol Salt Agar (Figure 1). Table 1: Differential characteristics of the genus Staphylococcus. Organism S. aureus S. epidermidis S. saprophyticus Coagulase positive negative negative Novobiocin Susceptibility sensitive sensitive resistant Mannitol Fermentation positive negative variable α-toxin Production positive negative negative

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Figure 1: The selective and differential medium, Mannitol Salt Agar. This medium is selective for salt-tolerant organisms, because it contains 7.5% NaCl and differential because the fermentation of mannitol in the medium results in a lowering of the pH and a change in the color of the pH indicator, phenol red, from reddish-pink to yellow. Both organisms are salt tolerant, but only the organism on the right is a mannitol fermenter.

Streptococcus Members of the bacterial genus Streptococcus and its close relatives in the genus Enterococcus are often found as part of the normal flora of the pharynx (upper throat). Because these normal flora are more varied, more involved biochemical testing is used to make a species identification. Typically though, inoculation of a mixed culture or patient sample onto blood agar will result in streptococcal or enterococcal colonies that are very small and grayish-green in color and may or may not exhibit hemolysis (Figure 2). This hemolysis is the result of the production of exotoxins which break down red blood cells in order to liberate iron and other materials for the use of the bacterial cell. It is important to remember that hemolysis refers not to the color of the colonies, but the color of the red blood cells in the medium. Colonies can be confirmed as Streptococcus or Enterococcus by Gram staining. After confirming the morphology of the cells as Gram positive cocci, other biochemical characteristics will allow for a genus and species identification (Table 2).

Figure 2: Hemolysis of red blood cells contained in sheep blood agar. Each organism is inoculated onto the plate in the shape of the Greek letter which identifies its hemolysis. Alpha (α) hemolysis results in the partial hemolysis of red blood cells adjacent to the growth of cells which is seen as a green “halo.” Beta (β) hemolysis results in the complete hemolysis of red blood cells and a yellow “halo” adjacent to cellular growth. Gamma (γ) hemolysis is simply a lack of hemolysis.

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Table 2: Differential characteristics of various members of the genera Streptococcus and Enterococcus

Organism Berg

ey’s

Grou

p

Lanc

efie

ld

Grou

p

Hem

olys

is

Baci

trac

in

Sen

siti

vity

CAM

P Re

acti

on

Hip

pura

te

Hyd

roly

sis

SX

T S

ensi

tivi

ty

Bile

Esc

ulin

H

ydro

lysi

s

Tole

ranc

e to

6.

5 %

NaC

l

Opt

ochi

n S

ensi

tivi

ty

S. pyogenes A β S - - R - - R

S. agalactiae B β R + + R - - R

S. pneumoniae n/a α R - - - - S

S. equi C β R - - S - - R

S. equisimilis C β R - - S - - R

S. zooepidemicus

Pyogenic

C β R - - S - - R

E. faecalis D α R - - R + + R

E. faecium Enterococci

D α R - - R + + R

S. bovis other D γ R - - + - R

S. mitis n/a γ R - - S - - R

S. salivarius n/a γ R - - S - - R

S. mutans

oral (viridans)

n/a γ R - - S - - R

Enterics Enteric (Greek enteron = intestine) bacteria are comprised of several different genera, but all reside in the digestive tract of mammals. Because the amount of bacteria in the large intestine particularly numbers in the billions, care must be taken to quickly eliminate all but the most obvious pathogens for routine medical care. Typically, enteric pathogens are Gram negative and lack the ability to ferment lactose. There are many commercially available media which are selective for Gram negative organisms and will differentiate them based on lactose fermentation. These media contain a dye that is taken up by lactose fermenters and will pigment these organisms, while lactose non-fermenters remain colorless. Eosin Methylene Blue (EMB) Agar is a medium in which the dyes eosin and methylene blue inhibit Gram positive bacteria. In addition, lactose fermenters will lower the pH of the agar, facilitating their uptake of the dyes, which causes them to display nucleated colonies with dark centers. MacConkey (MAC) Agar contains bile salts and crystal violet dye to inhibit the growth of most Gram positive bacteria and neutral red dye which stains lactose fermenters and provides the pigmentation associated with their identification (Figure 3). The characteristics that distinguish lactose fermenters from lactose non-fermenters are summarized in Table 3. Once the distinguishing characteristic of lactose fermentation has been determined, relatively few biochemical tests are necessary to determine the correct bacterial genus (Figure 4 and Figure 5).

Figure 3: Differential and selective media for the growth of Gram negative enterics, MacConkey (MAC) Agar and Eosin Methylene Blue (EMB) Agar. Media are selective for Gram negatives, by the addition of various dyes and chemicals and differential for lactose fermentation. Typically, lactose fermenters will

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appear pigmented on these so-called enteric media, and lactose non-fermenters will not. On MacConkey agar, lactose fermenters (left plate, two right side inoculations) appear dark pink. On EMB, lactose fermenters (right plate, two right side inoculations) appear purple or may have a metallic sheen, depending on the organism.

Table 3: Various Selective/Differential media for the isolation and characterization of Gram negative enteric bacteria.

Organism Eosin-Methylene Blue (EMB) agar MacConkey (MAC) agar Escherichia coli dark center with greenish metallic sheen red or pink

Enterobacter sp. similar to E. coli, but colonies are larger red or pink Klebsiella large, mucoid, brownish pink Proteus translucent, colorless transparent, colorless Pseudomonas translucent, colorless to gold transparent, colorless Salmonella translucent, colorless to gold translucent, colorless Shigella translucent, colorless to gold transparent, colorless

Figure 4: Dichotomous key for the identification of lactose non-fermenting enteric bacteria. Genus named listed in white will not be assigned to you as an unknown for this particular laboratory exercise. However, they may be assigned as a Major Unknown organism later in the semester.

Figure 5: Dichotomous key for the identification of lactose fermenting enteric bacteria.

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INTRODUCTION I. Staphylococci

In this experiment, you will be attempting to isolate and identify staphylococci from three sources: an unknown organism; your nose and a fomite. You will identify each of these as one of the three staphylococcal species discussed above. You will utilize several media to do this. First you will use selective and differential media which exploit the halotolerant nature of the genus Staphylococcus using m-staph broth, containing 10% NaCl and mannitol salt agar, which contains the fermentable sugar mannitol, the pH indicator phenol red and 7.5% NaCl. Additionally you will test your organisms for their susceptibility to the antibiotic novobiocin and determine whether or not your isolate produces α toxin, which causes β hemolysis. Finally, you may inoculate your organism into rabbit plasma, to test for the presence of the pathogenic enzyme coagulase (Figure 6).

Figure 6: Positive (upper) and negative (lower) results for coagulase production in rabbit plasma.

II. Streptococci

In this experiment, you will be attempting to isolate and identify streptococci from two sources: an unknown organism and up to two organisms from your pharynx. You will subject these isolates to several different selective and differential media to confirm the identity of each of these organisms. First, you will examine the hemolytic properties of your isolates using blood agar. Remember that hemolysins are exotoxins, and their effect will be seen in the media surrounding the colonies. Because your initial inoculation is onto differential and not selective media (like the staphylococcal protocol), you will need to select colonies that appear streptococcal. Streptococcal colonies are small because they are aerotolerant anaerobes; they are greenish-gray in color; and they may or may not cause hemolysis of the surrounding media. Once you have isolated putative streptococcal organisms, you will subculture each into tryptic soy broth (TSB) as a working stock. From there, you will test any α or γ hemolytic organisms for optochin susceptibility and salt tolerance using selective nutrient broth containing 6.5% NaCl. You will also inoculate your isolates into bile esculin agar (BEA) which is both a selective and differential medium. BEA contains 1% oxbile to inhibit non-group D streptococci and sodium azide to inhibit Gram negative bacteria. Organisms that are able to hydrolyze the esculin to esculetin will cause a precipitation reaction the ferric citrate Fe(C6H5O7)contained in the medium, resulting in a black precipitate (Figure 7). β hemolytic organisms are subcultured onto blood agar in order to determine whether the organism produces a CAMP factor which enhances the hemolytic capability of the isolate, resulting in an enlarged, arrowhead-shaped area of hemolysis when plated adjacent to Staphylococcus aureus. This CAMP test is inoculated perpendicularly to a line inoculation opposite a lawn on ½ of the blood agar plate upon which the antibiotics bacitracin and SXT are tested for susceptibility (Figure 8). Finally, organisms are tested for their ability to hydrolyze hippurate by adding the culture to a test tube containing hippurate and the indicator ninhydrin which detects the presence of glycine,

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produced as hippurate is hydrolyzed. This indicator is purple in the presence of glycine, and remains colorless when no glycine is present due to a lack of hippurate hydrolysis (Figure 9).

Figure 7: Negative and positive inoculations in bile esculin slants (BEA). Note that the medium is also selective and should inhibit any non-group D streptococci.

Figure 8: Inoculation onto blood agar for bacitracin and SXT susceptibility (upper half) and CAMP reaction (lower half) which results in an enhancement of its β hemolytic capability when inoculated perpendicularly to S. aureus (line inoculation along the bottom of the plate) seen as an arrowhead where the perpendicular lines of inoculation meet.

Figure 9: Positive and negative hippurate hydrolysis inoculations.

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III. Enterics

For this assignment, you will receive two individual broth cultures. One contains a lactose fermenting Gram negative bacillus and the other contains a Gram negative bacillus which lacks the ability to ferment lactose. It will be your job to determine the genus identity of both of these organisms using the biochemical inoculations outlined in the Protocol section of this Lab Exercise and the Dichotomous Key found in Figure 4. You will first inoculate these organisms side by side onto Eosin-Methylene Blue Agar (EMB) and MacConkey agar (MAC) to identify which is the lactose fermenter. You will then confirm this by inoculating your organisms into separate Lactose Phenol Red broth and a Glucose Phenol Red broth. Using these media, you will also be able to determine glucose fermentation. Both of these broths contain the sugar glucose or lactose and the pH indicator phenol red. These sugars may or may not be fermented by our organism. If the isolate is capable of fermenting it to produce an acidic byproduct and/or gas, which will be trapped in the inverted durham tube. If an organism ferments glucose only, the Glucose broth will by the only tube turns yellow. If an organism ferments lactose, both the Lactose and the Glucose broth will by the tubes should turn yellow. SIM or Kligler deeps contain ferrous sulfate (FeSO4), and if hydrogen sulfide (H2S) is being produced, it will react with the ferrous sulfate to form ferric sulfide (FeS2) (Figure 10). Because these tests rely on pH changes in the media, it is important to read the results in a timely manner. Once both glucose and lactose fermentation have been noted, you will test your lactose non-fermenter for motility and urea hydrolysis. Your lactose fermenter will be tested for indole production (using the multi-test medium SIM), urea hydrolysis and citrate utilization. As these media were used as part of your Minor Unknown, you should refer to Lab Exercise 18 for information about these tests and how to interpret them.

Figure 10: Various inoculations onto Kligler's Iron agar slants. Glucose fermentation is noted in the “butt” of the slant- a red butt indicates a lack of fermentation and a yellow butt indicates positive fermentation. Lactose fermentation is noted in the “neck” of the slant, with results similar to that of glucose: red necks are negative for lactose fermentation, yellow necks are positive. Gas production is noted as the slant either splits or is raised up from the bottom of the test tube. Inoculations which are capable of producing H2S form a black precipitate.

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PROTOCOL A summary of the media and results for all the staphylococcal, streptococcal and enteric inoculations can be found in Table 4. A diagram of the inoculations for the staphyloccocal inoculations can be found in Figure 11; a diagram of the streptococcal inoculations can be found in Figure 13 and a diagram of the enteric inoculations in Figure 15. I. Staphylococci Day One Protocol:

Individual Supplies 2 sterile cotton swabs Unknown control organism # ______ 3 m-staph broths

1. Using sterile technique, wet a cotton swab with your m-staph broth. Gently swab the lowest

portion of the inside of your nose. 2. Place the swab into the m-staph broth and label it accordingly. 3. Using a second tube of m-staph broth, and using sterile technique, wet a clean sterile cotton

swab. Swab the surface of any fomite in the lab. 4. Place the swab into the second tube of m-staph broth and label it accordingly. 5. Obtain an tube of an unknown Staphylococcus sp. Record unknown number. 6. Inoculate your Staphylococcal unknown into m-staph broth. 7. Incubate both tubes at 37°C for 48 hours.

Day Two Protocol: Individual Supplies 3 Mannitol Salt Agar plates Nose, fomite and unknown control m-staph broths

1. Using your inoculating loop, streak for isolation your fomite, nose and unknown organisms onto

separate Mannitol Salt Agar (MSA) plates. 2. Incubate the plates at 37°C for 48 hours. 3.

Day Three Protocol: Individual Supplies up to 1½ Blood Agar plates 1½ NA plates & antibiotic disk dispenser containing Novobiocin

1. Using Figure 1 as a reference, examine your MSA growth to determine salt tolerance and

mannitol fermentation for all three of your organisms. Gram stain all isolated colonies that appear different until you find one exhibiting morphology & arrangement indicative of staphylococcal bacteria. Only those organism which are confirmed as staphylococci should be further cultured and tested.

2. Organisms that ferment mannitol should be subcultured onto blood agar and inoculated into rabbit plasma to test for β hemolysis and coagulase production respectively.

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a. Rabbit plasma should be incubated in the 37°C water bath and checked every 30 minutes for coagulation, up to 24 hours.

3. All organisms showing growth on MSA should be checked for Novobiocin sensitivity using ½ NA plate for each isolate. Refer to Lab Exercise 13: Chemical Control of Growth for instructions on how to plate for antibiotic sensitivity.

Day Four Protocol:

1. Record all relevant data in your lab notebook. 2.

Figure 11: Inoculation diagram for the staphylococcal isolates.

II. Streptococci

Day One Protocol:

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Individual Supplies 1½ blood agar plate Unknown control organism # ______

1. Using a long sterile cotton swab, ask your lab partner to carefully swab your pharynx. 2. Spread this swab onto the primary zone of a Blood Agar plate. 3. Using your inoculating loop, streak the remaining quadrants for isolation. 4. Obtain an unknown control Streptococcal sp. Record the number. 5. Streak your unknown organism for isolation on ½ of a second blood agar plate. 6. Incubate the plates at 37°C for 48 hours.

Day Two Protocol: Individual Supplies up to 3 TSB broths

1. Using Figure 2 in the Introduction section, identify 2 colonies exhibiting α, β and/or γ hemolysis. 2. Inoculate each separately into TSB broth using your inoculating loop. 3. Incubate the tubes at 37°C for at least 48 hours.

Day Three Protocol:

Individual Supplies NA plates & antibiotic disk dispenser with Optochin Bile Esculin Slants 6.5% NaCl Blood Agar, S. aureus, Bacitracin and SXT incubated TSB & reagent kit for hippurate hydrolysis

1. Gram stain each of your isolates and confirm morphology and arrangement indicative of

streptococcal bacteria. 2. Using Table 2 in the Introduction section, determine the relevant inoculations for each of your

isolates growing in TSB. a. Optochin sensitivity testing can be done on ½ of a NA plate. b. Hippurate hydrolysis can be done directly from your TSB. c. Bacitracin and SXT sensitivity and CAMP testing must be done on Blood agar plates, and

media cannot be shared. See Figure 12 for a diagram of the inoculation procedure.

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Figure 12: Inoculation line of unknown organism for CAMP test and bacitracin & SXT susceptibility. Note that S. aureus is inoculated in a line perpendicular to the unknown organism.

3. With the exception of the Hippurate hydrolysis, all inoculations should be incubated at 37°C for

48 hours.

Day Four Protocol: 1. Record all relevant data in your lab notebook.

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Figure 13: Inoculation diagram for the streptococcal isolates.

III. Enterics Day One Protocol:

Individual Supplies Enteric A & Enteric B EMB agar plate MacConkey Agar plate

1. Streak your cultures on ½ of an EMB and MacConkey plate, as seen in Figure 14 below.

Figure 14: Inoculation lines for enteric A and enteric B on both EMB and MacConkey agar.

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Day Two Protocol:

Individual Supplies incubated EMB and MAC plates 2 glucose fermentation broths with Durham tubes 2 lactose fermentation broths with Durham tubes

1. Gram stain each of your isolates and confirm morphology and arrangement indicative of enteric

bacteria. 2. Tentatively determine which of the organisms is the lactose non-fermenter. 3. Inoculate both organisms into glucose and lactose sugar broths to confirm lactose non-

fermentation and check for glucose fermentation. 4. Incubate your broths appropriately.

Day Three Protocol: Individual Supplies Urea broth SIM Simmon’s citrate

1. Confirm the lactose non-fermenter, perform the following inoculations:

a. Identify your organism’s ability to ferment glucose. b. Inoculate your lactose non-fermenter into urea broth and SIM medium. c. Incubate your media at 37°C for 48 hours. Remember that urea broth is a 3–5 day

incubation and should be left in the incubator if a negative result is seen at the end of the 48 incubation period.

2. Confirm the lactose fermenter, perform the following inoculations: a. Inoculate your organism into SIM, Simmon’s citrate and urea broth. b. Incubate all media appropriately

Day Four Protocol:

1. Record all relevant data in your lab notebook.

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Figure 15: Inoculation diagram for the enteric isolates.

DATA AND OBERVATIONS I. Staphylococci

1. In a table similar to that below, record the results of each test performed in this experiment. Under Gram Stain indicate cellular shape/arrangement as well as Gram reaction. If you did not perform a test, indicate that with “n/a.”

Isolate Gram Stain Alpha Toxin Mannitol (Acid) Novobiocin

susceptibility Coagulase

Unknown No.____ Nose Isolate Fomite Isolate

2. Identify each of your staphylococcal isolates.

Unknown # ___: _________________________ Nose isolate: _________________________ Fomite isolate: _________________________

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II. Streptococci

1. Record here all information pertaining to the identification of your pharyngeal isolates and unknown. If you did not perform a test, indicate that with “n/a.”

Source of Unknown

Gram

sta

in

Hem

olys

is

Bacitr

acin

Sus

cept

ibili

ty

CAM

P Re

action

Hippu

rate

Hyd

rolysis

SXT

Sen

sitivity

Bile E

sculin

Hyd

rolysis

Toler

ance

to

6.5

%

NaC

l Opt

ochi

n Sus

cept

ibili

ty

2. Identify each of your streptococcal/enterococcal isolates. Unknown # ___: _________________________ Pharyngeal isolate 1: _________________________ Pharyngeal isolate 2: _________________________

III. Enterics

1. Complete the following table for each of your unknown organisms. If you did not perform a test, indicate that with “n/a.”

Biochemical Attribute Organism A Organism B

Gram stain lactose fermentation

glucose fermentation

motility urea H2S production indole citrate

3. Identify each of your enteric isolates.

Enteric A: _________________________ Enteric B: _________________________ DISCUSSION I. Staphylococci

1. Which tests are definitive for the identification of Staphylococcus aureus?

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2. Describe the selective and/or differential properties of mannitol salt agar (MSA) and m-staph broth for the isolation and identification of staphylococci.

3. What is the role of α-toxin in the pathogenesis of S. aureus?

4. What is the role of coagulase in the pathogenesis of S. aureus?

II. Streptococci

1. Differentiate between α-, β- and γ hemolysis.

2. What characteristics do Streptococcus pyogenes and Staphylococcus aureus share?

3. What tests would be useful for the differentiation of the two species?

4. Name three tests that are useful for the differentiation of S. pyogenes and S. agalactiae.

5. Name two tests that are useful for the differentiation of pneumococci and oral viridans streptococci.

III. Enterics

1. What additives make EMB and MacConkey agar selective and differential?

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2. What can be said of lactose non-fermenters on these types of media?

3. What two characteristics separate Salmonella from Shigella? What media can be used for this differentiation?

4. How can acid production by glucose and lactose fermentation be differentiated in the same tube?

5. What is alkaline reversion? Why is it important? Table 4: Various media and tests accomplished throughout the identification process for staphylococcal, streptococcal and enteric bacteria.

Staphylococcal Unknowns

medium biochemical/ physiological characteristic

enzyme reagent(s) positive results

blood agar hemolysis hemolysins (exotoxins) red blood cells (in medium)

medium indicates varying degrees of hemolysis by bacterial enzymes of the red blood cells within the medium; total hemolysis (β) is seen as a yellow halo around colonies; partial hemolysis (α) is seen as a green halo and no hemolysis (γ) is seen as no change in the medium

mannitol salt agar

7.5% salt tolerance; mannitol fermentation

fermentative lactose enzymes

phenol red (in the medium)

selective media upon which growth indicates halotolerance; yellowing of the medium indicates mannitol fermentation

m-staph broth

10% salt tolerance n/a none selective media within which growth indicates halotolerance

rabbit plasma coagulase production coagulase none coagulation of medium after incubation in 37°C water bath indicates presence of enzyme coagulase

Streptococcal Unknowns


enzyme reagent(s) positive results

6.5% NaCl broth

salt tolerance among streptococci

n/a 6.5% NaCl (in medium) growth indicates halotolerance

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bile esculin slant

organism’s ability to hydrolyze esculin, grow in the presence of oxbile and sodium azide

various 1% oxbile, sodium azide, esculin, ferric citrate (in medium)

no growth indicates susceptibility to oxbile and sodium azide (non-group D streptococci and Gram negatives); blackening of the slant indicates ability to hydrolyze esculin

blood agar hemolysis hemolysins (exotoxins) red blood cells (in medium)

medium indicates varying degrees of hemolysis by bacterial enzymes of the red blood cells within the medium; total hemolysis (β) is seen as a yellow halo around colonies; partial hemolysis (α) is seen as a green halo and no hemolysis (γ) is seen as no change in the medium

blood agar CAMP reaction CAMP factor (not an enzyme)

S. aureus, RBC (in medium)

organisms plated perpendicularly to S. aureus will have an arrowhead of hemolysis where the organisms meet, indicating the production of CAMP factor

TSB broth hippurate hydrolysis n/a hippurate & ninhydrin inoculation of culture into reagent tube with hippurate and added ninhydrin will cause a color change to purple

Enteric Unknowns


enzyme reagent(s) positive & negative results

Russell Double Sugar

multi-medium to test for sugar fermentation

fermentative lactose and/or glucose enzymes

[lactose] 10× [glucose] phenol red (in medium),

yellow butt indicates glucose fermentation, yellow neck indicates lactose fermentation,

Eosin-Methylene Blue agar

selective medium inhibits Gram positive; differentiates based on lactose fermentation

various eosin, methylene blue (in medium)

eosin & methylene blue are selective and inhibit Gram positive bacteria, are taken up by lactose fermenters resulting in pigmented and sometimes “nucleated” colonies; lactose non-fermenting organisms remain unpigmented

Kligler iron agar

multi-medium to test for sugar fermentation and H2S production

fermentative lactose and/or glucose enzymes, cysteine desulfurase

[lactose] 10× [glucose] phenol red (in medium), iron salts (in medium)

yellow butt indicates glucose fermentation, yellow neck indicates lactose fermentation, black precipitate indicates H2S production

MacConkey agar

selective medium inhibits Gram positive; differentiates based on lactose fermentation

various bile salts, crystal violet, neutral red (in medium)

bile salts & crystal violet are selective and inhibit Gram positive bacteria, neutral red dye is differential and pigments lactose fermenting organisms red; lactose non-fermenters are non pigmented

SIM H2S Production; indole production; motility cysteine desulfurase

iron salts (in medium); Kovac’s reagent

black precipitate indicates H2S production; red color change with Kovac’s reagent indicates indole production; cloudy throughout indicates motility

Simmon citrate agar

citrate as a sole source of carbon

citrase bromthymol blue (in medium)

blue color change indicates presence of citrase

urea broth production of urease urease phenol red color change to hot pink/cerise color indicates presence of urease

Lab Exercise: Staphylococcal, Streptococcal & Enteric...

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