Streptococcal Unknowns Miramar College

Biology 205 Microbiology Background 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 1). 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.

Figure 1: 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.

Lancefield Groups are from Rebecca Lancefield (January 5, 1895 – March 3, 1981) She was a prominent American microbiologist. She joined the Rockefeller Institute for Medical Research in New York in 1918 and was associated to that institute throughout her long and outstanding career. Her main achievements lie in her work on group A streptococci and its association with rheumatic fever. She is most famous for her serological classification of beta-hemolytic streptococcal bacteria which based on the carbohydrate composition of bacterial antigens found on their cell walls. The Lancefield classification system is still used internationally today.

Table 1: Differential characteristics of various members of the genera Streptococcus and Enterococcus

Organism Berg

ey’s

G

roup

Lance

field

G

roup

Hem

oly

sis

Baci

traci

n

Sensi

tivity

CAM

P

React

ion

Hip

pura

te

Hydro

lysi

s

SXT

Sensi

tivity

Bile

Esc

ulin

H

ydro

lysi

s

Tole

rance

to

6.5

%

NaCl

Opto

chin

Sensi

tivity

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

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 (throat). 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 2). 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 3). Finally (optional and may not be necessary), organisms could 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, 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 4).

Figure 2: 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 3: 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 4: Positive and negative hippurate hydrolysis inoculations.

Protocol: Streptococci

Day One Individual Supplies 1½ blood agar plates 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. Streak your unknown control organism for isolation on ½ of a second

blood agar plate. Share this plate with a lab partner. 5. Incubate the plates at 37°C for 24 - 48 hours. Day Two Individual Supplies up to 3 TSB broths

1. Using Figure 1 in the Introduction section, identify organisms 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 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 1 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 5 for a diagram of the inoculation procedure.

Figure 5: 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 24 - 48 hours.

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

Figure 6: Inoculation diagram for the streptococcal isolates.

Data Collection and Analysis 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

Gra

m s

tain

Hem

oly

sis

Baci

tracin

S

usce

pti

bilit

y

CA

MP

R

eact

ion

Hip

pu

rate

H

yd

roly

sis

SX

T S

en

siti

vit

y

Bil

e E

scu

lin

H

yd

roly

sis

Tole

ran

ce t

o

6.5

% N

aC

l

Op

toch

in

Su

sce

pti

bilit

y

2. Identify each of your streptococcal/enterococcal isolates. My unknown number _________ is _________________ ______________ My throat sample contains _______________ _________________ Discussion Questions: 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.

Media Used in Streptococcus analysis

Streptococcal Unknowns

medium biochemical/ physiological characteristic

enzyme reagent(s) positive results

6.5% NaCl broth

salt tolerance among streptococci

n/a 6.5% NaCl (in medium)

growth indicates halotolerance

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