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LABORATORY 2

Staphylococcus spp., Streptococcus spp., Enterococcus spp.

Objectives: Upon successful completion of this laboratory, you will be able to:

1. Demonstrate the cultural and biochemical characteristics used for identification of the species Staphylococcus, Streptococcus, and Enterococcus.
2. Perform a throat swab, a culture, and a rapid screening test for Group A streptococci.
3. Improve your skills at performing the Gram stain and at obtaining isolated colonies on agar media.
4. Identify a bacterial unknown using some of the tests currently employed in diagnostic microbiology laboratories.
5. Learn the basis of some of the rapid, miniaturized diagnostic methods used to identify Gram-positive cocci.

• Culture for colony isolation: Complete as directed.

1. Staphylococcus spp.— general

– Perform a catalase test to differentiate staphylococci from streptococci and enterococci.

– Perform a Gram stain on staphylococci.

2. Differentiate between S. aureus and other potential pathogenic Staphylococcus spp.

3. Differentiate between S. epidermidis and S. saprophyticus.

4. Streptococcus spp.- general

– Perform a Gram stain on Group A Streptococcus.

– Observe types of hemolysis (a, b, g) on BAP; observe surface and subsurface hemolysis.

5. Group A Streptococcus

• Observe bacitracin susceptibility and SXT resistance by Group A Streptococcus.

• Swab a throatand perform a rapid screening test for Group A streptococci

• Culture a throat for Group A streptococci

6. Group B Streptococcus

– Perform latex agglutination test for grouping streptococci, using Group B Streptococcus.

7. S. pneumoniae vs. viridans streptococci

8. Observe biochemical tests that differentiate between Streptococcus and Enterococcus spp.
9. Unknown No. 1, a Gram-positive

– Perform a Gram stain, catalase test, and other tests as indicated.

1. Examine the plates that you streaked for isolated colonies (from "Demonstration Broth # 1, 2, 3, 4, or 5" on agar plates) to see if you got well-isolated single colonies. If not, see your group instructor for help with your four-phase streaking technique.

Can you detect two colony types on any of the plates?

2. Note the colony morphology, color, and hemolysis on BAP plate.

Colony-type	Observations on BAP
	Morphology	Color	Hemolysis
1	.	.	.
2	.	.	.

The aerobic gram-positive cocci most commonly associated with infection include members of the genera Staphylococcus Streptococcus, and Enterococcus. The enterococci were previously considered Group D streptococci. Quite often, the site of the lesion and a smear of the exudate are beneficial in making a tentative diagnosis. However, such presumptions are dependent on knowledge of the normal flora for any given site. There are many similarities among members of these genera, and their extracellular metabolic products provide the key to laboratory identification and differentiation of the various pathogens.

The main criterion for differentiating between Staphylococcus and the other two species are the catalase test.

Most aerobes and facultative anaerobes possess the enzyme, catalase, which enzymatically releases O₂ from hydrogen peroxide (H₂O₂). The catalase test (O₂ release from 3% H₂O₂) is a useful laboratory diagnostic tool for the differentiation of Staphylococcus, which are catalase positive, from Streptococcus and Enterococcus spp., which are catalase negative.

Divide a microscope slide into 4 sections with a glass marking pencil. Transfer a small portion of a well-isolated colony of S. aureus from the BAP onto one section of the slide. Add one drop of 3% hydrogen peroxide (use small vials labeled "catalase reagent") to that area. Observe for ebullition of gas (effervescence). Do not dig into the BAP, since erythrocytes contain catalase: this could yield a false-positive reaction.

Similarly test S. epidermidis, S. saprophyticus and any one of the streptococci supplied on a separate BAP. Only the staphylococci should be catalase-positive. What is the biological importance of catalase to those organisms producing it?

Staphylococci are common residents of normal skin. They cause most suppurative infections of the skin, but they also produce severe infections in any other part of the body. Colonies of staphylococci on solid media are soft, round, opaque, and vary in color from cream to orange. Smears from these colonies may show the characteristic grape-shaped clusters or may appear singly or in short chains resembling streptococci.

Do a Gram stain on either S. aureus or S. epidermidis (or both if you like!), using the method you learned in lab #1.

Observe the stained smears for gram-positive cocci. Note the cellular arrangement. Ask an instructor to examine your slide and critique your staining technique.

There are at least three species of the genus Staphylococcus of clinical importance: S. aureus, the most pathogenic for humans, S. epidermidis, which is part of the normal flora and is of low pathogenicity, and S. saprophyticus which can cause urinary tract infections, especially in sexually active young women. The production of coagulase by S. aureus is the single most useful criterion for differentiating it from other species. In addition, most strains of S. aureus are pigmented, produce hemolysis on blood agar plates, ferment mannitol, and produce DNase. As the latter properties vary from isolate to isolate, they are not as reliable a differential aid as coagulase production.

Observe the BAP of S. aureus and S. epidermidis for hemolysis. Note the beta-hemolysis (clear zone) for S. aureus and no hemolysis for S. epidermidis. (S. saprophyticus is also non-hemolytic). Note that not all strains of S. aureus are beta-hemolytic and some non-pathogenic species of staphylococci are beta-hemolytic.

Note the pigment of S. aureus streaked on a BAP. The colonies of most strains are pigmented, ranging from cream yellow to orange. Approximately, one-half of S. saprophyticus strains are also pigmented, ranging from cream yellow to yellow-orange. S. epidermidis is usually white.

The ability to clot plasma is the most widely used and generally accepted criterion for the identification of S. aureus. The test for production of the enzyme coagulase may be done with either oxalated or citrated rabbit plasma that is commercially available. The most accurate method employs 0.5 ml of plasma to which is added several colonies from a plate or 0.5 ml of broth culture. The plasma tube is covered with parafilm to prevent evaporation and incubated at 37^oC. Once a coagulum, no matter how small, has formed the test is considered positive. The plasma should be incubated overnight before a test is called negative, but prolonged incubation (over 24 hours) may result in the dissolution of the clot. Pathogenicity of the organism is related neither to the rapidity nor the amount of coagulation, merely its presence or absence. In some laboratories, the staphylococci that are negative by the tube coagulase test are collectively referred to as coagulase-negative staphylococci.

Observe the demonstration at each bench showing positive and negative coagulase production. This test is read by slowly tilting the tube and observing for the highly viscous clot formation in the plasma. A negative test results in the plasma remaining free flowing with no evidence of a clot.

There are several rapid diagnostic methods for identifying S. aureus in the modern diagnostic microbiology lab, including rapid tests for coagulase. In general, the rapid tests detect the presence of one or more proteins that are secreted by S. aureus or that are on the surface of the S. aureus bacteria. One test, the Staphyloslide, detects the presence of a fibrinogen receptor and/or Protein A on the S. aureus surface. The fibrinogen receptor, also known as clumping factor, can bind to soluble fibrinogen (or to a fibrin clot). Fibrinogen is one of several serum proteins that coat our own cell surfaces and also deposit onto implanted devices (e.g., catheters). The ability of S. aureus to adhere to fibrinogen is thought to promote staphylococcal colonization of tissues and implanted foreign bodies. The presence of the fibrinogen receptor distinguishes S. aureus from other staphylococci and is thought to contribute to the virulence of S. aureus. Protein A is found on the cell surface of about 95% of S. aureus isolated from humans and has the ability to bind the Fc portion of immunogloublin G (IgG). The Staphyloslide test consists of blue latex particles coated with human fibrinogen and IgG. Colonies of staphylococci that have clumping factor or Protein A on their surface will bind the latex particles causing a visible agglutination of particles. S. aureus will cause notable agglutination. Agglutination will not be seen if the isolate is S. epidermidis or S. saprophyticus.

In practice, if this rapid test gives equivocal results or results that conflict with other findings for the isolate, the conventional coagulase test with rabbit plasma is performed.

Observe an instructor performing the Staphyloslide test, using cells of S. aureus and S. epidermidis from isolated colonies on a BAP.

There are 2 reagents to use for each bacterium to be tested: the tube labeled Test Latex contains latex particles coated with human fibrinogen and IgG, the tube labeled Control Latex contains uncoated latex particles.

1. Use the reaction card provided.
2. Tap the tubes gently several times to resuspend the latex; if using a dropper, expel any latex from the dropper for complete mixing.
3. Add 1 drop of Test Latex onto one of the circles on the reaction card and 1 drop of Control Latex onto another circle.
4. Add 1 colony of the Staphylococcus being tested to each drop. Mix well with your loop to cover the circle.
5. Rock the slide manually 20 seconds. Read for agglutination immediately under normal lighting conditions.
6. A positive test for S. aureus will show clumping with the Test Latex within 20 seconds and none with the Control Latex. A negative reaction will show no clumping within 20 seconds for either reagent.

Limitations of the Procedure:

The test is uninterpretable if the Control Latex shows agglutination. This may occur with some strains of staphylococcus that have been incubated longer than 36 hours.

Some streptococci and other organisms that posses immunoglobulin or plasma binding factors on their surface may react with the Test Latex and some strains of E. coli non-specifically agglutinate latex particles. Therefore, it is important that a Gram stain be performed before the Staphyloslide so that only staphylococci are tested.

S. saprophyticus, once considered a contaminant, is an important cause of urinary tract infections in sexually active young women and in older men who have indwelling catheters. S. saprophyticus is resistant to novobiocin (at a concentration of 5 mg), and this is used to differentiate the organism from S. epidermidis which is susceptible to novobiocin.

Observe the demonstration plate (Plate A-2e) streaked with S. saprophyticus on one half and S. epidermidis on the other half. There should be a zone of inhibition greater than 16 mm around the novobiocin disc placed on the S. epidermidis growth and a zone less than or equal to 16 mm around the disc placed on S. saprophyticus.

In some clinical laboratories, staphylococci identification is aided by the use of commercially supplied sets of miniaturized biochemical tests or nuclei acid tests. However, first, as always, isolated colonies must be obtained on primary plating media. Representative colonies are noted for their morphology, tested for their Gram reaction, catalase activity and the stained cells observed for morphology and arrangement just as you have been doing today. This information, coupled with the information from the physician about the source of the specimen determines the sets of miniaturized tests that need to be carried out. These tests are all inoculated reproducibly and rapidly, often in a single action. Nonetheless, they still take time to produce results, because many of the tests depend upon metabolic utilization of substrate and growth of the bacteria to a sufficient degree to register as a significant finding. The physician has to bear this in mind when submitting specimens to the diagnostic lab.

Some laboratories choose to restrict complete species identification of the coagulase negative staphylococci to isolates from normally sterile sites (such as blood, joint fluid, or cerebrospinal fluid) or to routinely distinguish S. saprophyticus from other coagulase-negative staphylococci only if isolated from urine.

Streptococci are the third most common group of organisms seen in clinical laboratories, exceeded only by the Enterobacteriaceae and the staphylococci. Enterococci are a prominent cause of nosocomial infections and are intrinsically resistant to many antibiotics. Many of these organisms are commonly found as part of normal human flora but can cause life-threatening infections if accessible to normally sterile sites.

Microscopically streptococci and enterococci are are round or oval-shaped, and may occasionally be elongated. They are gram-positive and may occur singly, in pairs, in short chains, or in long chains, depending upon specific and/or type of culture or specimen. In sputum, pus, serous fluids, and liquid cultures, these gram-positive cocci may be indistinguishable in appearance from each other. Streptococcus pneumoniae, the pneumococcus, are generally lancet-shaped diplococci (i.e., paired, ovoid cocci with their adjacent ends flattened). In direct smears, a capsule may be observed surrounding the pneumococcus.

Perform a Gram stain on each of the following: S. pyogenes, S. pneumoniae and S. mitis (a viridans streptococcus) obtained from the BAPs at your bench. Each of the samples can be applied to different sections of one slide or prepare separate slides containing one sample each.

Note the cellular arrangement. Can you detect any significant difference in morphology between these species?

Although streptococci and enterococci have a much different colonial morphology from the staphylococci, both of these genera of bacteria are gram-positive cocci that could conceivably be confused when viewing the Gram stain. To differentiate between the staphylococci and the streptococci and enterococci, a catalase test is performed before determining which tests to use to confirm and speciate staphylococci, streptococci, or enterococci.

The various streptococci have traditional genus-species Latin names, and you will encounter these designations. However, traditionally, clinical laboratories report them by their type of hemolysis and Lancefield serological group (e.g., beta Streptococcus-Group A). The first step in correctly identifying a Streptococcus in the clinical laboratory is an accurate determination of the type of hemolysis the organism produces. Streptococci may be divided into three groups on the basis of their hemolytic action on blood agar:

Study the hemolysis demonstration plate (plate B-2a) at your bench.

Recognize the three types of hemolysis, a , b , and g .

More than 99% of all b -hemolytic streptococci isolated from humans can be placed in the Lancefield serological groups A, B, C, F, or G, with Group A being by far the most common. Since Group A strains are the most pathogenic for humans, it is important to differentiate them from the other beta-hemolytic streptococci.

Some b -hemolytic streptococci are poor surface hemolyzers. Therefore, a plate must be stabbed 3-4 times, as well as streaked, in order to detect b -hemolysis. Two hemolysins are responsible for b -hemolytic activity in Group A streptococci. These are differentiated by antigenicity and susceptibility to inactivation by oxidation. Some strains of Group A streptococci may have only one of the hemolysins.

One half of a demonstration plate (Plate B-2b) has been inoculated with a Group A Streptococcus which exhibits only streptolysin O activity. This hemolysin is oxygen-labile (hence, the name, streptolysin "O"); and b -hemolysis is demonstrated only by the subsurface colonies, which are under reduced oxygen tension. The surface colonies appear a -hemolytic as a result of the inactivation of the streptolysin O by oxygen. This demonstrates why a BAP must be stabbed when attempting to identify a Group A b -hemolytic Streptococcus. The other half of the BAP has been inoculated with a Group A Streptococcus that has only streptolysin S, which is oxygen stable, hence the name streptolysin "S". Since this hemolysin is not inactivated by oxygen, b -hemolysis is demonstrated by both surface and subsurface colonies.

At the UKMC Diagnostic Microbiology Laboratory, b -hemolytic colonies typical of streptococci are initially typed for Lancefield group specific carbohydrates using commercially available kits. However in some laboratories, a single b-hemolytic colony typical of streptococci is streaked on a fresh BAP, and a bacitracin disc is placed, aseptically, on the inoculated area of the first streak quadrant prior to incubation. (A pair of forceps dipped in alcohol and then flamed can be used to aseptically transfer a disc from the vial to the culture plate.) A zone of growth inhibition indicates the presence of Group A b -hemolytic streptococci. Many a -hemolytic streptococci, including pneumococci, are susceptible to bacitracin when a small inoculum is used. Therefore, hemolysis must be read correctly before calling a bacitracin-susceptible organism a Group A Streptococcus.

Although the bacitracin susceptibility test is 95% accurate for recognizing Group A streptococci, rare strains of Groups B, C, D and G may show some degree of bacitracin susceptibility. To avoid incorrect identification of b -hemolytic streptococcus as Group A, a disc containing the sulfonamide, sulfamethoxazole and trimethoprim (SXT) is used in conjunction with a bacitracin disc. For convenience, the SXT and bacitracin susceptibility testing may be done in the same inoculated area of the plate. Group A streptococci are resistant to SXT. If the b -hemolytic organism being tested is susceptible to both bacitracin and SXT, it is not Group A.

Observe the demonstration plate (Plate B-2c) streaked with Group A Streptococcus. There should be a zone of inhibition around the bacitracin disc, but no inhibition around the SXT disc. Note the placement of the discs: they are about 2 cm apart from each other and placed within the heavily inoculated (undiluted) part of the plate, away from the edge.

In this exercise, we will perform a latex agglutination test (Streptex) that is used by the Diagnostic Microbiology Laboratory. The test identifies Groups A, B, C, D, F, and G streptococci by antibodies to the Group-specific carbohydrates. These Group-specific carbohydrate antigens can be released from the bacterial surface by treatment of the bacteria with a proteolytic enzyme. Not all the Lancefield antigens are specific for a single streptococcal species; however, the presence of group B antigen seems to correlate with a strain’s identity as S. agalactiae, which is an important cause of serious neonatal infection.

In the Streptex test, group-specific antibodies are coated onto polystyrene latex beads. When the latex beads are incubated with a proteolytic extract containing the corresponding released Group-specific carbohydrate antigen, a strong antigen-antibody reaction occurs, that crosslinks the beads in a clearly observable agglutination reaction.

Divide into groups of 4, one student will perform the Streptex test for Group B Streptococcus (GBS) using the following procedure:

1. Using a tube containing 0.4 ml of extraction enzyme solution, make a light bacterial suspension using your loop and the BAP streaked with Group B Streptococcus. 5 large colonies should do, or make a single sweep across the growth on the plate.
2. Incubate in the 37^oC waterbath for 5 min.
3. Shake the tube to mix.
4. Incubate 5 min. more (can go as long as 45 more min.).
5. Set out the tube containing the extracted Group B streptococci and set out a Reaction Card for the antigen-antibody reaction.
6. Mix the latex-antibody suspensions well by shaking vigorously for a few seconds. Holding the dropper bottles vertically, dispense 1 drop onto a circle on the Card for each test performed. A positive and negative control should be also be tested to assure that all reagents are working properly.
7. Using a Pasteur pipette, place 1 drop of extract into one latex-antibody-containing circle. Place 1 drop of the positive control and one drop of a negative control into separate circles containing latex-antibody.
8. Mix the liquids in each circle with a mixing stick or toothpick, using a fresh stick for each suspension. Spread the liquid to cover the whole circle area. (Discard the mixing sticks into the Lysol jugs on top of the bench.)
9. Gently rock the Card for no longer than 1 minute and then read the results. You should see a strong agglutination reaction for your extract only in the circle containing the Group B-latex reagent. (Discard the Reaction Cards in the biohazard containers in your lab sector).

Due to the non-suppurative sequelae of Group A steptococcal pharyngitis (notably rheumatic fever and acute glomerulonephritis), several rapid screening methods have been developed for use in the physician’s office for the detection of Group A antigen in throat specimens. The sensitivity of each method depends on the numbers of streptococci present in the sample and the particular method used. In general, a throat swab obtained from a patient suspected of having Group A streptococcal pharyngitis is placed in an extraction solution to release any Group A antigen present in the specimen. After a short incubation, any Group A antigen in suspension is detected by a variety of technologies. These detection methods are rapid and specific. However, specimens containing small numbers of bacteria may produce false-negative results.

On each bench is a protocol for the performance of a rapid screening test for Group A streptococci from a throat swab. Read the protocol and make note of the factors that may alter the test results. A positive control test will be available for observation.

Each student has a specimen that was obtained over the weekend, labelled with a number from 1-100, and stored at 4⁰C. Using a rapid screening kit, perform a test for the detection of Group A streptococcal antigen. Discard the swab in biohazard waste. Consult with an instructor if you do not get a positive result.

Divide into groups of two and culture the posterior pharynx (over the tonsillar area) of your partner (see figure). USE ONLY the STERILE swabs and tongue depressors provided.

Avoid touching the tongue, the lateral walls of the buccal cavity, or the uvula.

Inoculate a BAP by rubbing the swab back and forth over the upper sixth of the plate. Using a flamed loop, distribute the inoculum over one third of the plate. Without flaming the loop, stab the loop into several areas containing the inoculum to distribute any beta-hemolytic streptococci below the agar surface (WHY?). Flame the loop and streak the plate for colony isolation. Label appropriately and place on a tray for incubation.

Group D streptococci may exhibit a -, g -, or occasionally, b -hemolysis. They may be differentiated from other streptococci by their ability to hydrolyze esculin in the presence of bile salts; all Group D organisms will grow and blacken the medium.

Observe the demonstration tubes at each bench showing positive and negative reactions for esculin hydrolysis.

Enterococci may also exhibit b-hemolysis. All strains of Enterococcus hydrolyze esculin and strains usuually isolated from humans produce the Group D streptococcal antigen. In the clinical laboratory, identification of enterococci specieis requires performing a variety of biochemical tests or nucleic acid testing. In these labs, use the following chart to presumptively identify the strains of streptococci and Group D enterococci.

Presumptive Identification of b-hemolytic streptococci and enterococci

^aSymbols: +, positive reaction or susceptible; -, negative reaction or resistant; *, exceptions occasionally occur.

^bSXT - sulfamethoxazole and trimethoprim.

The non-beta hemolytic streptococci include the S. pneumoniae, viridans streptococci, Group D streptococci and enterococci.

The a -hemolytic streptococci, or viridans (green) group (e.g. S. mitis, S. sanguis, S. mutans, S. salivarius), are normal inhabitants of the mouth, nasopharynx, and respiratory tract. Most of these species can, with some difficulty, be placed into one of the Lancefield groups, but this is not commonly done. Alpha-hemolytic streptococci may be isolated by blood culture in cases of bacterial endocarditis. When found in other clinical specimens, they generally are not the etiology of the disease.

S. pneumoniae, a leading cause of bacterial pneumonia and meningitis, may harmlessly inhabit the upper respiratory tract. On BAP, pneumococcal colonies are surrounded by a greenish zone of a -hemolysis. NOTE: S. pneumoniae (pneumococcus) requires elevated CO₂ concentrations for growth. Therefore, isolation of this organism requires incubation in 5% CO₂ or a candle jar for growth. Young S. pneumoniae colonies are round, shiny, gray, and domed. As the culture ages, autolysis allows the central portion to sink, yielding a flattened surface or central depression. Metabolically, pneumococci resemble the other streptococci, but they possess autolytic enzymes that cause the lysis of the organisms in old cultures (and the central depressions of colonies). As a result, in older cultures, some pneumococci may stain Gram-negative. Surface-active agents, such as sodium deoxycholate, activate the pneumococcal autolytic enzymes and lead to dissolution of a colony or to loss of turbidity in a suspension. These agents rarely lyse other streptococci; hence, the bile-solubility test is a rapid way to differentiate S. pneumoniae from alpha hemolytic streptococci or enterococci.

One student in a group of 4 or 5: Place a drop of 10% sodium deoxycholate directly on an isolated colony of S. pneumoniae. Keep the plate level to prevent the solution from running across the plate and leave at room temperature until the reagent dries (no more than 15 minutes). Pneumococcal colonies will disappear or flatten, while viridans streptococci will be unaffected.

The Optochin disc is used in most laboratories for differentiation. Discs impregnated with optochin (ethylkhydrocupreine hydrochloride), when placed on a freshly inoculated blood agar plate, will inhibit the growth of pneumococci but allow other a -hemolytic streptococci to grow normally. When colonies suspected as pneumococci are seen, several colonies are streaked on a fresh blood agar plate and an Optochin disc is placed on the inoculated area of the first streak quadrant prior to incubation (as was done for bacitracin and SXT discs for the Group A Streptococcus). The plate is then incubated for 18-24 hours.

A zone of inhibition 14 mm or greater is read as a positive test; a zone of 6 to 14 mm is a questionable result.

Observe the demonstration plate for Optochin susceptibility of S. pneumoniae (Plate B-2f).

Perform a Gram stain on S. pneumoniae and a viridans streptococcus obtained from the BAP at your bench. Note the cell morphology and arrangement.

Once S. pneumoniae has been ruled out as a possibility for an alpha-hemolytic colony, viridans streptococci, Group D streptococci and enterococci must be considered.

Enterococci are a part of the normal flora of the human gastrointestinal and urinary tract. The patient’s own endogenous flora is the source of MOST infection with this organism. Urinary tract infection is the most common human enterococcal infection. Enterococci can be differentiated from Group D streptococci by the ability of enterococci to grow in 6.5% NaCl broth.

One to two colonies of suspected isolates of enterococci were inoculated into 6.5% NacCl broth and incubated overnight at 37⁰C..

Observe the visible turbidity in the broth containing enterococci compared to the uninoculated broth.

For this course, use the following table to aid in the identification of non-beta-hemolytic streptococci and Group D enterococci.

____________________________________________________________________________

^aSymbols: +, positive; -, negative. Strains of Group B streptococci (S. agalactiae) may be non-hemolytic; latex agglutination with Grp B antigen should be used to rule out possible group B strains.

^bOccasional strains of viridans streptococci produce weakly positive bile esculin reactions.

____________________________________________________________________________

Unknown No. 1 consists of a single species from one of the three genera of bacteria that were demonstrated in the laboratory today; the organism may be either a pathogen or member of the normal flora. The unknown will be handed out during the second hour of laboratory; this will give you time to observe the demonstrations. It will come to you already streaked out on agar medium commonly used for the organism. You will use a subset of the demonstrated media and biochemical tests in the identification of your unknown.

• Blood agar plates
• Streptex
• Staphyloslide
• Bile-solubility test
• Bacitracin, SXT, and Optochin discs
• Bile-esculin slants

1. Record your unknown number.
2. Record colony morphology and type of hemolysis on BAP.
3. Perform a Gram stain on your unknown. Record the gram reactivity, cell morphology and arrangement.
4. Perform a catalase test. Record the results.
5. Perform Staphyloslide or Streptex if indicated. Record your results.
6. Streak plates and add appropriate discs, if indicated. Label all plates properly and place on trays for incubation. If you
7. Place your original unknown plate in the box labelled "Unknown cultures" located in the front of the room.
8. When you have completed the identification of your unknown (either during this lab or in Lab 2), place your completed Unknown No. 1 sheet in the appropriate box in the lab.

1. What antibiotic therapy would normally be appropriate?
2. Is there a vaccine to prevent or control infection due to this organism?
3. What other bacterial pathogens, if any, might possibly cause similar disease?
4. How frequently is the identified pathogen associated with clinical disease?

NAME _______________________________ DATE_____________________

A. Colonial morphology, color, and hemolysis on BAP

B. Gram stain results (circle one): positive, negative, gram-variable

Cell morphology and arrangement:

 

C. Catalase results (circle one): positive or negative

D. Presumptive identification of organism:

E. Further tests performed for confirmation and results.

G. If results are inconclusive, what other tests are needed to identify the organism?

GRADE: ________________ (TOTAL POSSIBLE, 0.5 points)

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