ExerciseⅥ The ldentification of Pathogenic Coccus
Materials
Staphylococcus aureus；S.epidermidis；S.saprophyticus；Streptococcus pneumoniae；Streptococcus equi（Group C）；tryptic soy broth culture of Streptococcus mitis（Group D）；tryptic soy broth culture of Streptococcus pyogenes（Group A）；tryptic soy broth culture of S.agalactiae（Group A）；S.staphylococcus mannitol salt agar plate；3% hydrogen peroxide（H2 O2）（caustic）；blood agar plate；hydrogen peroxide；novobiocin disks；polymixin disks；SXT disks（Difco）；Bacitracin disks；c AMP disk；bile esculin agar slants；Bunsen burner；inoculating loop；Gram-staining material.
Learning Objectives
Each student should be able to
1.Recognize the medical significance of the staphylococci.
2.Perform selected clinical procedures that distinguish the pathogenic species of staphylococci from the nonpathogenic species.
3.Perform biochemical and serological diagnostic tests to identify pneumococci and differentiate them from other hemolytic isolates.
4.Perform several biochemical and serological tests to identify S.pyogenes.
Principles
1.Staphylococcus
The genus Staphylococcus consists of gram-positive cocci，0.5 to 1.5μm in diameter，usually in irregular clusters，within which pairs and tetrads are commonly seen（Figure 1）.They are nonmotile and nonsporing.Members of this genus are facultatively anaerobic.Colonies are round，convex，mucoid，and adherent to the agar.They are chemoorganotrophic，requiring nutritionally rich media.Staphylococci have respiratory and fermentative metabolism，producing acid but no gas from carbohydrates（Figure 2）.They are able to grow on nutrient agar with 5% NaCl and are usually positive for catalase.Optimum growth is at 37℃.Staphylococci are commensals on the skin and in the human mouth and upper respiratory tract.They can be human pathogens.
Figure 1 Morphology of Staphylococci
Gram's stain smear prepared from a staphylococcal culture illustrating the characteristic grapelike clusters of gram-positive cocci（×1，000）Corresponding book color chart NO.52.
Figure 2 Cultures of Staphylococcus aureus on VogeiJohnson Agar
V-J agar is a modified tellurite-glycine agar containing phenol red indicator and mannitol that is selective for recovery and identification of coagulase positive S.aureus.S.aureus grows on this medium and produces gray to black colonies from the production of free tellurium.A yellow halo is typically seen around the colonies from the fermentation of mannitol and the production of an acid p H.Tellurite，lithium chloride，and high glycine content inhibit the growth of many grampositive and gram-negative bacteria.NO.53 S.aureus（L.aureus，golden）is the most important clinical member of this genus.It may be isolated from the skin or mucous membranes of the body.It can cause various infections（e.g.，carbuncles，abscesses，pneumonia，endocarditis，food poisoning，toxic shock syndrome）throughout the body.
S.epidermidis is a nonpathogenic member of this genus（Figure 3）.It is part of the normal microbiota of the skin.Additionally，S.saprophyticus can be isolated and may be responsible for urinary tract infections.
Figure 3 Culture of Staphylococcus epidermidis and S.aureus（L.aureus，golden）on Blood Agar
Notice the white，opaque，nonhemolytic，smooth colonies characteristic of S.epidermidis.Corresponding book color chart NO.54.
Clinical tests for staphylococci are directed toward（1）separation of the genus Staphylococcus from other gram-positive cocci（such as Micrococcus or Streptococcus）and（2）separation of the species within the genus.Table 1 lists some of the characteristics used to separate members of this genus from the other genera.Table 2 lists characteristics used to differentiate between the three most commonly isolated Staphylococcus species.
Table 1 Some Characteristics Used to Distinguish Gram-Positive Cocci
Table 2 Some Characteristics of Staphylococcus Species
continued table
One of the most popular ways to isolate and identify S.aureus and other gram-positive cocci of medical importance is through the use of blood agar plates.Hemolysis patterns are particularly useful in identification.Inα-hemolysis，a zone of greenish coloration with an indistinct margin forms around colonies growing on blood agar（Figure 2）.The color results from partial decomposition of hemoglobin.β-hemolytic is a sharply defined zone of clear hemolysis with no greenish tinge surrounding the colony（Figure 4）.Of course，some gram-positive cocci such as S.salvarius produce no hemolysis on blood agar.
Figure 4 Culture of Staphylococci on Blood Agar NO.55
Blood agar plate on which large，smooth，β-hemolytic colonies of S.aureus are growing.
Mannitol salt agar also can be used for the selectiveisolation，cultivation，and enumeration of staphylococci from clinical and nonclinical specimens.Mannitol-using bacteria（e.g.，S.aureus）turn the medium yellow（Figure 5），whereas those that do not use mannitol（e.g.，S.epidermidis，S.saprophyticus）produce no color change（Figure 6）.
Figure 5 Culture of Staphylococcus aureus on Mannitol Salt Agar NO.56
Notice that the medium has turned yellow around the growing bacteria since the bacteria are able to ferment the mannitol and produce an acid p H.
Figure 6 Culture of Staphylococcus epidermidis on Mannitol Salt Agar NO.57
Notice the small white colonies that do not use the mannitol；that is，no color change is observed since no acid has been produced.
2.Pneumococci
S.pneumoniae（pneumococci）is the causative agent of many types of diseases（e.g.，bacterial lobar pneumonia，conjunctivitis，otitis media，meningitis，peritonitis）.Thirty to seventy percent of normal individuals harbor this bacterium in their pharynx.S.pneumoniae is a gram-positive coccus（the distal ends are lancetshaped），usually arranged in pairs or short chains（Figure 7）.It also occurs singly.The pneumococci are somewhat fastidious in their nutritional needs and do not survive well in competition with other microbiota.The medium of choice for culturing pneumococci is blood agar（Figure 8）.Colonies that form on this medium appear small and shiny with a dense center and a lighter，raised margin，giving them a dimpled appearance.The hemolytic pattern observed on blood agar is α-hemolysis（a zone of greenish coloration occurs around the colonies）.
Figure 7 Morphology of Pneumococci NO.58
Gram stain of a direct smear illustrating several short chains and many gram positive Streptococcus pneumoniae diplococci clumped together（×1000）.
More presumptive tests for pneumococci are bile solubility and optochin tests.In the bile solubility test，pneumococci will dissolve in bile（2%to 10%sodium deoxycholate），whereas alpha streptococci will not.In the optochin test，the pneumococci exhibit sensitivity to optochin（ethylhydrocupreine）and will lyse，but alpha streptococci are unaffected（Figure 8）.
Figure 8 Streptococcus pneumoniae on Blood Agar NO.59
Notice the zone of inhibition around the optochin disk.This indicates sensitivity，a characteristic useful in distinguishing S.pneumoniae from otherα-hemolytic streptococci.
3.Streptococci
The streptococci are gram-positive cocci arranged in chains.Streptococci are classified according to their hemolytic activity，immunologic properties，and resistance to chemical and physical factors.
Streptolysins produced by S.pyogenes cause the lysis of red blood cells in vitro，producingβ-hemolytic（a clear zone of hemolysis with no color change）on blood agar（Figure 9）.Two types of beta lysins are produced：streptolysin Oand streptolysin S.
Figure 9 Streak Plate of Streptococci on Blood Agar NO.60
This blood agar plateillustrates the small，semitranslucent graywhite colonies of streptococci surrounded by zones ofβ-hemolytic.
Pyogenes is classified as Group A，based on a chemical substance known as C carbohydrate（an antigenic，group-specific hapten）found in the cell wall.In addition to hemolysis，S.pyogenes can be distinguished from viridans streptococci by means of the bacitracin sensitivity test.In this test，a filter paper disk impregnated with 0.04 units of bacitracin is applied to the surface of a blood agar plate that has been previously streaked with the bacteria to be identified.The appearance of a zone of inhibition surrounding the disk is a positive test for Group A streptococci（Figures 10，11）.An absence of a zone of inhibition suggests non-Group A bacteria（e.g.，Group C）.
Figure 10 The Group A and Group C Streptococcal Responses to Bacitracin and SXT
Figure 11 Bacitracin Sensitivity Test NO.61
The right half of this blood agar plate was inoculated with a pure culture of a Group Aβ-hemolytic streptococcus.The left half of the plate was inoculated with Streptococcus mitis.The two disks contain 0.04μg bacitracin.After 24 hours of incubation，the inhibition of growth around the antibiotic（disk on the right）is a positive bacitracin test.The bacteria on the left are bacitracin resistant.(https://www.daowen.com)
Group B streptococci can be distinguished from otherβ-hemolytic streptococci by their production of a substance called the CAMP factor.CAMP is an acronym for the names of the investigators（Christie，Atkins，and Munch-Petersen）who first described the factor.This factor is a peptide that acts together with the β-hemolysin produced by some strains of S.aureus，enhancing the effect of the latter on a sheep blood agar plate（Figure 12）.
Figure 12 The CAMP Test for Distinguishing Group B Streptococci from Otherβ-hemolytic Streptococci.
Group D streptococci and enterococci can be differentiated from other streptococci by using bile esculin agar slants.Group D streptococci grow readily on the bile esculin agar and hydrolyze the esculin，imparting a dark brown color to the medium.This reaction denotes their bile tolerance，ability to hydrolyze esculin，and constitutes a positive reaction.Gram-positive bacteria（other than group D streptococci and enterococci）are inhibited by the bile salts.
This exercise is designed to familiarize the student with the morphological and colonial characteristics，diagnostic tests（Table 3.1），and serological identification of S.pyogenes.
Table 1 Diagnostic Test for Streptococcal Differentiation
Procedure
1.Gram stain
First we do the gram stain.Most coccus are gram positive，some are gram negative，for example，the Neisseria，including the N.meninggitidis and N.gonorrhoeae.
2.Catalase Activity
（1）To test for catalase，pipette several drops of a 3%solution of H2 O2 over the growth on the glass slide
（2）Picked some growth from a slant with inoculating loop and then mix in the H2 O2
（3）The appearance of gas bubbles indicates a positive test；the absence of gas bubbles is a negative test（Figure 13）.
Figure 13 Catalase Test on Slides
A positive catalase reaction（left slide）shows gas bubbles；a negative catalase reaction reveals an absence of gas bubbles（right slide）.
Result：Staphylococcus（catalase positive），Streptococcus（catalase nagative）
For Staphylococcus，Select one presumptive colony of S.aureus，S.epidermidis and S.saprophyticus for further characterization
3.Catalase Activity
（1）Add 0.5 ml of citrated rabbit plasma to two small test tubes.With the wax pencil，label the tubes with the respective bacteria，your name，and date.
（2）Inoculate one tube with enough S.aureus paste to make a cloudy suspension.Inoculate the other tube with S.epidermidis and S.saprophyticus.
（3）Incubate both tubes at 35℃for 1 to 4 hours in a water bath.Afterward，examine both tubes for the presence or absence of clouding and clots.A positive test（coagulase activity）is indicated by the formation of a clot（Figure 14）.
Coagulase producing strains of S.aureus form a clot（solid fibrin gel）when grown in plasma（tube on the left），whereas coagulase negative staphylococci[S.saprophyticus（middle tube）and S.epidermidis（tube on the right）]do not form a clot.
Result：S.aureus positive，S.epidermidis and S.saprophyticus negative
4.Fermentation of mannitol
1）Seed the presumptive colony of S.aureus，S.epidermidis and S.saprophyticus in the culture containing phenol red indicator and mannitol.
2）Inoculate the tubes under 37℃for 18 to 24 hours.
S.aureus will form small，black colonies surrounded by a yellow zone due to the fermentation of mannitol.It will form large golden colonies surrounded by wide yellow zones.
S.epidermidis and S.saprophyticus will form smaller black colonies with no yellow zone due to the inability to ferment mannitol.These colonies may even be surrounded by a deep red color.On a mannitol.
Figure 4.2 Coagulase Test
Result：S.aureus positive，S.epidermidis and S.saprophyticus negative
5.Susceptibility testing
（1）Use a separate，sterile cotton swab for each bacterium.The swab is immersed in the culture tube，and the excess culture is squeezed on the inner side of the test tube.
（2）The swab is then taken and streaked on the surface of the plate three times，rotating the plate 60°after each streaking.Finally，run the swab around the edge of the agar.This procedure ensures that the whole surface has been seeded.Allow the culture to dry on the plate for 5 to 10 minutes at room temperature with the top in place.
（3）Dispense the antibiotics（novobiocin disks and polymixin disks）onto the plate either with the multiple dispenser or individually with the single unit dispenser.Make sure that contact is made between the antibiotic disk and the culture by gently pressing the disk with alcohol-flamed forceps.
Do not press the disk into the agar，and do not move the disk once it is placed on the agar.
（4）Incubate the plates for 16 to 18 hours at 35℃.do not invert the plates.
Result：Novobiocin is sensitivity and polymixin is resistance of S.epidermidis，Polymixin is sensitivity and polymixin is resistance of S.saprophyticus.
For Streptococcus，Select one presumptive colonies of，A，B，C，D group and Pneumoniae.
6.Hemolysis
Blood agar plate.Incubate the plates for 16 to 18 hours at 37℃.Observe for pigmentation and hemolysis.Pneumoniae observed on blood agar isα-hemolysis（a zone of greenish coloration occurs around the colonies）.A，B group of Streptococcus areβ-hemolytic.The bile solubility test can also be carried out on isolated colonies.Optochin-impregnated disk also be proceed to confirm the Pneumoniae.
Result：Pneumoniae isα-hemolysis.
7.Bacitracin and SXT sensitivity test
（1）inoculate one blood agar plate with Group A，S.pyogenes and another plates with Group B（S.agalactiae），C（S.equinus）Use a loop to inoculate.
（2）.Place a bacitracin disk and an SXT disk on the plates.Be sure to place the disks on opposite sides of the plates，in well-inoculated areas.Incubate the plate for 24 hours at 35℃.Do not invert.
Result：Group A is bacitracin sensitive，Group C is SXT sensitive.
8.CAMP and esculin hydrolyzation test
（1）With an inoculating loop，streak a strain of S.aureus down the center of a blood agar plate.On one side of the plate，inoculate a strain of Group B streptococci（S.agalactiae）by making a streak at a 90°angle，starting 5 mm away from the S.aureus and extending outward to the edge of the agar.On the other side of the plate，inoculate the strain of Group A streptococci（S.pyogenes）.This streak should not be directly opposite the Group B streak.
（2）Incubate the plates at 35℃for 24 hours.
（3）Inoculate unknown bacteria with medium containing esculin，Incubate the slants at 35℃for 24 hours.
（4）Observe the area of hemolysis around the S.aureus streak.At the point adjacent to the streak of Group B streptococci，you should be able to see an arrowhead-shaped area of increased hemolysis indicating the production of the CAMP factor.There should be no change in the hemolytic zone adjacent to the streak of the Group A streptococci since most strains do not produce the CAMP factor.
（5）Examine the esculin agar slants for any color change.A dark brown color change is a positive reaction for group D streptococci.No color change is a negative reaction.
Result：Group B produces of the CAMP factor.Group D is esculin positive.
HINTS AND PRECAUTIONS
（1）Be sure to carefully measure the diameter of inhibition zones at the proper time to get accurate results.
（2）It should be noted that the zone of hemolysis around the bacitracin disk is so distinct because a pure culture is being used.If this is not so，then your stock culture is probably contaminated.
（3）Sometimes it is easiest to observe hemolysis by examining the zone with the 4 X objective of a compound microscope.
