STREPTOCOCCUS

 There are a wide variety of species included in the genus Streptococcus, many of which are innocuous to humans.  There are those, however, which are important agents of infection.  Particularly significant as human pathogens are S. pyogenes, S. pneumoniae, and Enterococcus. fecalis.  Several others produce disease in humans, though they are more commonly associated with lower animals.

 The members of the Streptococci are gram-positive organisms that usually form pairs or chains. Further classification is quite complex because of their wide variation, distribution, and effects.  In 1937, Sherman separated the genus according to physiological and growth characteristics, especially with regards to temperature limitations on growth.  His four general groups are (1) pyogenic, (2) viridans, (3) enterococcus, and (4) the lactics.  This categorization has become somewhat obsolete as relationships between species have been shown to overlap.

 The Streptococcus  may also be designated according to the type of hemolysis they produce on blood agar.  Thus (1) alpha-hemolytic, (2) beta-hemolytic, or (3) gamma-hemolytic (or nonhemolytic) streptococci are commonly used designations.

 Lancefield developed a serological classification based on the presence of C substance, an extractable carbohydrate found in the cell wall. At present, there are at least 20 groups, each of which is separable from the others by serology.  Each is given a capital letter (A, B, C,etc.) designation, of which Group A is most pathogenic in humans.  A given serologic group may or may not have  more than one species to a group (e.g., Group A contains only S. pyogenes, but Group D includes Ent. fecalis, S. bovis, S. equinus, S. avium, and S. faecium).

 Consequently, nomenclaturally the organisms in this genus may be referred to in several ways.  These are by the (1) type of hemolysis they produce,  by the (2)Lancefield group,   by the (3)Sherman classification, or   by their (4) formal species designation.

 Antigenic Group, Habitat, and Some Infections Caused
by Some Selected Members of the Streptococci

Species                 Antigenic Group           Usual Habitat in Humans                                                     Infections
 

S. pyogenes                      A                          Throat, respiratory tract                                 Pharyngitis, wounds, pneumonia,
                                                                                                                                                      scarlet fever, impetigo, etc.

S. agalactiae                     B                           Intestinal, genital, urinary tracts                 Urinary tract, wounds, otitis media,
                                                                                                                                                      infant (meningitis,septicemia)
                                                                                                                                                      diseases

S. equi                                C                           Upper respiratory tract                                 Impetigo, pharyngitis

Ent. fecalis                         D                           Feces                                                              Endocarditis

S. pneumoniae                 -                             Respiratory tract                                           Pneumonia, otitis media,
                                                                                                                                                      meningitis, septicemia, etc.

S.  mutans               (Viridans)                        Oral microbiota                                            Endocarditis, tooth decay

MATERIALS:

A.   Per table:

 Stock culture of: Streptococcus pyogenes, S. agalactiae, Ent. fecalis,   S. mutans, and
S. pneumoniaeradially streaked on a blood agar plate,
 plus a culture of Staphylococcus aureus  ATCC 25923
 12 - Sheep blood agar plate (SBA)
 1 - Bile esculin agar plate (BEA)
 Alcohol beaker with forceps
 Optochin discs (ethylhydrocupreine hydrochloride)
 Bacitracin discs
 Sterile swabs
 Tongue depressors
 Bile solution (sodium desoxycholate reagent)

LABORATORY PROCEDURES:

Part 1.    WEDNESDAY.

A.    KNOWN CULTURES.

Many of the organisms used here are primary pathogens.
BE CAREFUL.   PLAN YOUR WORK BEFORE YOU START.

  Each table will be provided with stock cultures of Streptococcus pyogenes, S. agalactiae, Ent. fecalis, S. mutans, and S. pneumoniae.  Working in a group of two.

 1.  Prepare a gram stain of each organism.

  NOTE:  Observe for differences in morphology, length of chains, and in some instances the ovoid nature of the sphere etc.  Also, compare these observations with some of those previously made on the staphylococci  (e.g., lines of symmetry and perfect spheres).

 2.  Streak for isolation each organism on one-half of a sheep blood agar  plate (the group needs three plates for the five organisms). Also stab each organism into the agar using the loop (at approximately a 45 degree angle) in other words,  puncture the agar a couple of times in an unused portion of the streaked sector.

NOTE: The stab is usually done after the first sector streak, before flaming the loop.  This stabbing will allow for subsurface growth leading to reduced amounts of oxygen.  Since one of the hemolysins, Streptolysin O, produced by S. pyogenesis inactivated by oxygen, consequently, such growth conditions will accentuate the hemolytic activity of this organism.

 3.  Inoculate  the 5 species to each of five sectors on a:

  a.   Sheep Blood Agar (SBA) plate to determine the effects of optochin and bacitracin on the growth of these organisms.  Use a heavy inoculum, streaking the entire sector for confluent growth placing one species per sector of the plate. Using alcohol flamed forceps, aseptically place a bacitracin differentiation disc in one area of each of the sector.   Additionally, place an optochin disc in each of the same sector, but in an area well away from the bacitracin discs.  Gently press each disc with the forceps so as to make good contact with the agar.

  b.  Bile esculin agar (BEA) plate using a radial  streak (one straight line inoculation) of each of the five organisms. This medium contains oxgall, an bile product and esculin, a glycoside along with a ferric salt.  The function of each is explained later.

 4.   Perform the CAMP assay as follows:

  a.  Make a single heavy straight  line streak of Staphylococcus aureus  ATCC #25923 across the center of a blood agar plate.  Next, make a straight line streak of  each of the five streptococcal species, perpendicular and up to the S. aureus  streak, but not through it.   Place two of the streptococcal species on one side and the other three on the other side.  Make sure there is ample space between the strep streaks.

 5.   Incubate all cultures for 24 hours as follows:

  a.  All blood agar plates at 37OC in a candle jar (a closed container that has a lighted candle allowed to burn to extinction to increase atmospheric CO2 concentration, but does not produce anaerobic conditions).   The SBA with throat cultures (#3 below) will also be incubated in the candle jar.

  b.  The BEA plates in the  37O C incubator without augmentation.
 
 

Part 2.   WEDNESDAY.

A. THROAT CULTURES.

   The species of Streptococcusare common parts of the normal microbiota of the naso- and oropharyngeal cavity, as well as other areas of the host.  Therefore, pathogenic streptococci must be differentiated from among these. Streptococcus pyogenesisolation is aided by the use of blood agar plates with stabs to allow subsurface growth, giving improved conditions for this organism to give its typical beta-hemolysis.    S. pneumoniaedoes not  require this condition to produce its characteristic alpha-hemolysis.

 1.  Swab the nasopharyngeal area of your throat or ask your partner to swab your throat.

 2.  Inoculate the surface of the SBA plate by rolling the throat swab over a small portion of the agar surface.  Streak for isolation with a loop.

 3.  Incubate your SBA plate for 24 hours at 37O C in a candle jar.
 
 

Part 3.   THURSDAY. OBSERVE RESULTS.
 

A.    THE KNOWN ORGANISMS:

 1.  Blood agar plates for isolated colonies:

  Observe for the presence of (alpha, beta or gamma) hemolysis, colonial morphologic features, and other characteristics of the 4 streptococcal species employed.  Also, note differences in surface and subsurface hemolytic patterns obtained on sheep blood agar.

 2.  Blood agar plates for chemical sensitivity:

  Compare (by measurement) the zone of inhibition of growth due to bacitracin and optochin.

  Bacitracin.

  a. Any inhibition of growth by bacitracin is considered positive.

  Optochin.

  a. The inhibition zone size due to optochin depends on the diameter of the disc.  If the disc is 10 mm in diameter, an inhibition zone of 16 mm or more is considered positive; if it is 6 mm, a zone size of 14 mm or more is positive.

   NOTE:  Group A strep or S. pyogenesis sensitive to bacitracin.  Whereas, S. pneumoniaeis sensitive to optochin.

 3.  Bile esculin agar plate:

  Bile:

  This medium contains a bile product known as oxgall, and if anything grows, that means that it is bile tolerant.   In general, most microorganisms are bile sensitive and will not grow.

  a. Observe for growth which indicates a bile tolerance.

  Esculin.

  a. Observe a dark color around the growth which indicates esculin hydrolysis.

  NOTE:  The hydrolytic product reacts with the ferric compound in the medium yielding a gray or black color.

  NOTE:  All Group D streptococci are bile tolerant and hydrolyze esculin.

 4.  Bile solubility test:

  Carry out the bile solubility test on an isolated colony directly on the SBA plate.  Do so by placing a drop of bile solution (sodium desoxycholate reagent) on a colony.  Allow to stand for 5 minutes, then tilt the plate to drain the excess solution to the side of the plate.  Observe for the presence or disappearance of the colony by reflected light.

  NOTE: The basis for the test:  The bile solution activates an autolytic amidase (autolysin) that cleaves the bonds (between D-alanine and muramic acid) in the peptidoglycan  resulting in lysis of the organism.

  NOTE:  The pneumococci are positive (the colony dissolves and therefore considered to be bile soluble).

 5.   CAMP test:

   A positive test is seen as an enhanced hemolysis, usually in an “arrowhead” pattern, at the junction of the streak of this organism with that of S. aureus.

  NOTE: Neither organism produces a clean clear beta hemolysis on their own.  However when combined, the result is a clear complete hemolysis.  The hemolysin of S. agalactiaeand the staphylococcal beta-lysin (sphingomyelinase) produce a synergistic effect of complete hemolysis.

  NOTE:  This test is positive with Group B strep or S. agalactiae.

 6.  Tabulate your results.

Bring out distinguishing characteristics of each.

 7.  Save the known cultures on a blood agar plate divided into a dozen or more sectors.  This should include all the “strep” cultures and also the “staph” ATCC culture.   The unused sectors are for the throat isolates from the following section.  Incubate the plate overnight at 37O C,  thereafter store at room temperature in the assigned cabinet  until next Wednesday.

B.   THE THROAT ISOLATES:

 NOTE: The wide variety of colonies that are present on the throat isolation plate.  The normal throat may be the habitat for many bacterial species, including gram negative cocci as Neisseriaand Moraxella; gram-negative rods of the genus Haemophilus and occasionally the coliforms; and gram-positive diphtheroid bacilli of the genus Corynebacterium.

 NOTE:  After performing some microscopic examinations, do any of these types of organisms seem to be present on your plate?

 1.  Select several typical alpha and beta-hemolytic (streptococcal) colonies.

  a.  Alpha hemolytic colonies:

    S. pneumoniaeis usually seen to form alpha-hemolytic, small, flattened (i.e. effuse), transparent surface colonies with a moist surface. As the pneumococcal colonies become older the colony center collapses giving a cratering  effect.

NOTE:Compare the colonies of the known  S. pneumoniae with suspected colonies on throat culture plate.

   i.   Describe the colonial characteristics.

   ii.   Prepare a gram stain of each.

  b.  Beta hemolytic colonies:

    i.  Describe the colonial characteristics.

   ii.  Gram stain each for cellular characteristics.

           2.   Make a stock of each isolate on a blood agar plate divided into multiple sectors.  This is the plate in instruction #7 above.  Incubate at 37O C, overnight.  Store at room temperature until Wednesday when it will be used.
 

Part 4.  The Following WEDNESDAY.

 DIFFERENTIAL INOCULATIONS.

 The cultures from last week have been saved on a multisectored blood agar plate.

 Each student will need a sheep blood agar plate divided into at least eight sectors.  Four for the knowns and the rest for your isolates

A.   ALPHA HEMOLYTIC THROAT ISOLATES:

 1.  Heavily inoculate each alpha hemolytic organism to a sector of a plate of sheep blood agar.  Place an optochin disc on the inoculated portion of the plate.*

 2.  Incubate  in candle jar at 37O C.

B.   BETA HEMOLYTIC THROAT ISOLATES:

 1.  Streak and stab your beta-hemolytic streptococcal isolate on to a sector of a SBA and apply a bacitracin disc.*

 2.  Incubate  in candle jar at 37O C.

 *Note:  Both the alpha and beta hemolytic isolates with the respective optochin and bacitracin discs can be done on a single plate.

 3.   On a seperate blood agar plate, carry out a CAMP test with any beta hemolytic isolate, sharing the plate with  your lab partner(s).  Be sure to include a S. pyogenes  and a  S. agalactiae control culture streak.  A plate of blood agar should accomodate about six organisms (three on one side and three on the other)  for testing.   Make sure there is ample space between the streaks.

 4.   Incubate  in candle jar at 37O C.
 
 

C. Possible Demonstrations:

  1.  Alternate rapid methods for serologic grouping of the Streptococcus are available.   These are an alternative to the tedious and involved method using the hot acid extract of the C polysaccharide from the cell wall.

   For example, the Phadabact Streptococcus Test (Pharmacia Diagnostics, Piscataway, N. J.) is designed to detect Groups A, B, C, and G by coagglutination.  This test uses Staphylococcus aureus  protein A as a binding site for the Fc portion of the antibody molecules directed against these groups. The Fab portion is then free to react with the respective group epitope on whole, untreated streptococci.    A positive test results in an agglutination-type reaction.

   A similar principle is employed for the Streptex Latex Agglutination Test (Wellcome Reagents Limited, Research Triangle Park, N.C.) except antibodies are attached to latex beads.  In this case, antigen is extracted with an enzyme.  It can detect Groups A, B, C, D, F, and G.

   The Serostat Streptococcus Test (Scott Laboratories, Inc., Fiskville, R.I.) also utilizes latex beads to detect Groups A, B, C, and G, but employs untreated broth culture organisms.
 

Part 5.  The Following THURSDAY.

 OBSERVE RESULTS - THROAT ISOLATES.

A.   ALPHA HEMOLYTIC THROAT ISOLATES:

 1.  Examine SBA cultures for the hemolytic activity and growth characteristics and describe each.

 2.  Optochin test.

  a.   Measure the zone of inhibition.  Refer to the requirements as discussed previously.

 3.  Bile solubility test.

        Carry out only if optochin positive.

  a.   Place a drop of bile (sodium desoxycholate) solution on the edge of the growth.

  b.   Allow to stand for 5 minutes, then drain excess solution to the side of the plate.

  c.   Observe for the presence or disappearance of the growth by reflected light.

 NOTE:  Pneumococci are alpha hemolytic, optochin sensitive, and bile soluable.  However,  any alpha hemolytic optochin resistant, bile insoluable organism is generally considered a viridan streptococci.

 Question:   Is your isolate pneumococcus or is it of the viridans group?
 
 

B.  BETA HEMOLYTIC THROAT ISOLATES:

 1.   Record hemolytic characteristics .

 2.  Bacitracin test.

  a.   Any amount of zone inhibition is considered a positive test.

  NOTE:   Group A or S. pyogenes  is bacitracin positive.

 3.  CAMP test.

  a.  Read the results given by your isolate and by the control culture.

 Question:  What can you conclude if anything concerning the species name of your isolate(s)?