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1. Demonstrate cultural and biochemical characteristics used for identification of species of Neisseria.
2. Demonstrate cultural and biochemical characteristics used for identification of species of Haemophilus.
3. Know the requirements for the growth of N. gonorrhoeae, N. meningitidis, and Haemophilus spp.
4. Understand the value of a gram stain for the diagnosis of genital gonorrhea.
5. Perform test for b -lactamase production.
6. Describe the concept for the Kirby-Bauer method for antibiotic susceptibility testing of bacteria.
7. Analyze the use of and need for minimal inhibitory concentration (MIC) determinations in testing bacterial susceptibility to antibiotics.
8. Understand the concept of the E-test method for generating a MIC value.
9. Improve your skills at performing the Gram stain and at obtaining isolated colonies on agar medium.

 Sequence of Student Work and Observations

1. Conclusion of Laboratory 2
   
   – Throat culture for Group A streptococci: Complete as directed.
   
   – Unknown No. 1: Complete as directed.
2. Neisseria spp.
   
   – Observe gonococci in prestained urethral exudate.
   
   – Perform a Gram stain on Neisseria sicca.
   
   – Observe differential effect of Thayer-Martin medium.
   
   – Observe oxidase test on N. sicca and S. epidermidis.

– Observe sugar fermentation tests for speciation of the genus Neisseria.

3. Haemophilus spp.
   
   – Perform a Gram stain on Haemophilus influenzae.
   
   – Observe nutritional tests for speciation of the genus Haemophilus.
4. Antibiotic Susceptibility Testing
   
   – Perform a Cefinase rapid test for detection of b-lactamase production.

2. Unknown No. 1

Two pathogenic species of Neisseria may be encountered in clinical specimens, and the source of a Gram-negative diplococcus may be indicative of the identity of the organism. N. meningitidis (meningococcus) is frequently found in the nasopharynx of normal individuals and is the cause of dramatic epidemics of cerebrospinal meningitis. N. gonorrhoeae (gonococcus, GC) is found in association with gonorrheal infections, one of the most prevalent of the sexually transmitted diseases. Both of these species require enriched medium (Chocolate Agar [CAP] or Thayer-Martin [T-M] medium) and increased CO₂ for growth. They are usually difficult to maintain on artificial media for prolonged periods of time. Other members of the genus Neisseria that occur as normal saprophytes on mucous membranes and sometimes on the skin are rarely pathogenic and are not fastidious in growth requirements.

N. meningitidis has the capacity of autolysis; thus, smears and cultures from spinal fluid or other specimens should be made immediately, as complete autolysis of the organisms may occur within 30 minutes. Often, diagnosis of meningococcal meningitis must be made on the basis of a correctly performed Gram stain in the emergency room. Cultures of this Gram-negative diplococcus often will not grow because of autolysis or because of prior antibiotic treatment of the patient before hospitalization.

The presence of intracellular, Gram-negative diplococci in a male urethral smear is usually sufficient for tentative diagnosis of gonorrhea and the beginning of treatment. However, it is not the case with smears from females because non-pathogenic, Gram-negative diplococci normally inhabit the genital area.

Scan the Gram-stained smear of a urethral exudate (in the slide box at the center of each bench), using your oil immersion objective, for intracellular, Gram-negative, kidney-shaped diplococci having adjacent flattened sides.

Please—After you're done, blot off the immersion oil gently and return the slide to the box for future use.

Perform a Gram stain on Neisseria sicca from the CAP. Observe the stained smear for Gram-negative diplococci and sketch one or two typical pairs of cells.

Because the gonococcus is delicate and is easily killed by drying, the assessment of cure must be accomplished by culture, which should preferably be made at bedside or in the clinic. The greatest number of gonococcal cultures are positive when specimens are inoculated directly onto a nutritive growth medium such as modified Thayer-Martin and incubated immediately at 35-37⁰C in 3-10% CO₂. University Hospital's Diagnostic Microbiology Laboratory will inoculate, also, BAP and CAP for primary plating analysis of specimens suspected to contain gonococci or meningococci. T-M medium contains antimicrobial agents (vancomycin, colistin, and nystatin) which inhibit the growth of normal flora, including nonpathogenic Neisseria, and permit a presumptive identification of the gonococcus to be reported in 18-24 hours. The medium itself has increased concentrations of agar and glucose, with trimethoprim lactate added to inhibit contaminating organisms such as Proteus species. When delays in transport are expected, systems that incorporate culture medium and a method for increasing CO₂ are required. The JEMBEC plate (an acronym from John E. Martin Biological Environmental System) is one type of system in use today. It contains modified Thayer-Martin medium and a molded inner well that holds a CO2-generating tablet.

Observe the Thayer-Martin Medium plate and note the good growth of N. gonorrhoeae (or N. meningitidis) versus inhibited growth of the nonpathogenic N. sicca (Plate A-2a).

Demonstration A-1c. Oxidase Test for the Neisseria:

The oxidase reaction is based upon the production of an enzyme, indophenol oxidase (cytochrome oxidase), by members of the genus Neisseria. The only other oxidase-positive organisms which are likely to occur in the same culture as Neisseria belong to the genus Pseudomonas and are Gram-negative bacilli. The reaction is employed in most laboratories on suspected cultures of gonococcus or meningococcus. A drop of freshly prepared tetramethyl-p-phenylenediamine dihydrochloride or p-aminodimethylaniline oxalate is placed directly on suspected colonies. The development of a deep purple color indicates a positive reaction. If a colony is to be transferred to another medium, it may be done within 10 to 15 minutes after addition of the reagent because the organisms are not immediately killed.

To be performed by instructors: Perform the oxidase test by adding a drop of oxidase reagent to a colony of N. gonorrhoeae grown on chocolate agar. Observe for a positive reaction. Repeat the oxidase test using a S. epidermidis culture and observe for a negative reaction. This test is performed with colonies from primary isolation plates; the results help determine which multi-test system to inoculate for further identification.

1. observing growth on plain nutrient agar (non-pathogens are not fastidious)
2. by using nucleic acid probes
3. by acid production from carbohydrates

 Traditionally, CTA (Cystine Trypticase agar) medium with 1% carbohydrates added was used for detection of acid production from carbohydrates but its use is no longer recommended. Several commercial multitest systems are available which reliably detect acid production and other biochemical tests within several hours of incubation. In the UKMC Diagnostic Microbiology Laboratory, if the miniaturized multitest system gives a result that conflicts with other information on the isolate or if the result is equivocal, the CTA sugar fermentation tests will be performed. A fluorescent antibody method is used to confirm identification of isolates from children or in sexual abuse cases.

Tube A illustrates a positive test--growth occurred and acid was produced from the sugar as denoted by the turbidity and yellow color, respectively, in the upper part of the medium.

Tube B shows a negative result as indicated by turbidity (growth), but no accompanying yellow color (acid was not produced from the sugar).

Tube C illustrates what happens if the culture being used is not pure; note the positive reaction involving the entire tube of medium. This indicates that the reaction is due to a contaminant or, if the culture is pure, that the organism being tested is not a species of Neisseria.

Differentiation of Neisseria species

1. Haemophilus spp.

Members of the genus Haemophilus are minute Gram-negative coccobacilli. H. influenzae is found in the respiratory tract of humans and can cause meningitis in children. When present in spinal fluid, these organisms are frequently pleomorphic, being seen as rods, coccobacilli, or thread-like forms. Four species of Haemophilus are commonly found in the pharynx of normal individuals: H. influenzae, H. parainfluenzae, H. hemolyticus, and H. parahemolyticus. Of these, in general, only H. influenzae produces serious infections: epiglottitis (croup), laryngitis, pneumonia, meningitis, etc. H. ducreyi causes chancroid—a venereal disease with lesions resembling the chancre of syphilis. Chancroid is rare in Kentucky.

Most of the species of Haemophilus can be characterized by a nutritional requirement for hemin (heat-stable X factor) and/or nicotinamide-adenine-dinucleotide (NAD, heat-labile V factor) which are found in blood. H. influenzae requires both X and V factors which are supplied in chocolate agar.

Observe the culture of H. influenzae growing on a CAP.

Note the luxuriant growth on this medium resulting in colorless, translucent, moist colonies.

Perform a Gram stain on H. influenzae obtained from the CAP at your bench.

Sketch two or three typical cells.

Haemophilus identification plates are available commercially from several manufacturers. These plates contain one quadrant each for X factor enriched medium (Quadrant I), V factor-enriched medium (Quadrant II), both X and V factors-enriched medium (Quadrant III) and horse blood to determine growth and hemolysis (Quadrant IV). Note that H. influenzae does not grow when only X factor or V factor alone is present. However, H. parainfluenzae does grow when only V factor is present indicating that only NAD is required for growth.

H. haemolyticus is occasional normal flora of the upper respiratory tract. It requires both X and V factors as does H. influenzae. However, H. haemolyticus shows a wide zone of beta hemolysis on horse blood agar, which differentiates it from H. influenza, which is not hemolytic on horse blood agar.

Observe the Hemo ID QUAD plate (Plate B-2a) showing growth of H. influenzae, which requires both X and V factors, only in Quadrants III and IV.

Observe the Hemo ID QUAD plate (Plate B-2b) showing growth of H. parainfluenzae , which requires only the V factor, in Quadrants II, III, and IV.

Certain bacteria are able to produce enzymes that inactivate b -lactam antibiotics. All of these enzymes share the capability of hydrolyzing the amide bond in the b -lactam ring found in all the b -lactam antibiotics (penicillins, cephalosporins, monobactams and penems) and, therefore, are classified as b -lactamases. In many instances, the production of one of these enzymes in a bacterium is predictive of resistance to b -lactamase-sensitive antimicrobial agents. Laboratory tests for rapid detection of b -lactamase production can be made after primary isolation, 18-24 hours prior to the time that growth-dependent susceptibility results would be available. This is especially helpful for the proper treatment of diseases such as meningitis, where time is of the essence. The three major bacterial causes of meningitis in two- to six-year-olds are S. pneumoniae, N. meningitidis, and H. influenzae—penicillin is a good drug for all of these. However, penicillin-resistance has forced the use of expensive b -lactamase-resistant cephalosporins or other alternative antibiotics. A rapid laboratory test for b -lactamase employs the Cefinase disc, which is impregnated with the chromogenic cephalosporin, Nitrocefin. This compound is yellow in its complete state but becomes red upon cleavage of the b -lactam ring. Therefore, when a bacterium produces b -lactamase in significant quantities, the yellow colored disc turns red on the area where the isolate is smeared within 5-10 minutes. A negative result will show no color change on the disc. Detection of b-lactamase activity is effective for H. influenzae, N. gonorrhoeae, Moraxella catarrhalis, Enterococcus spp., anaerobic bacteria, and Staphylococcus spp. The Diagnostic Microbiology Laboratory uses this test for these isolates.

At each bench is a BAP labeled Bla⁺ (b-lactamas-producer) and Bla^- (nonproducer of (b-lactamase). One person at each bench should perform this test.

Moisten the two Cefinase discs with one drop of tap water per disc, using a loop to transfer the water. Smear some Bla⁺ organism on one disc and some Bla^- organism on the other. Observe the positive and negative reactions for b-lactamase production. How long does it take to get a clear-cut difference for the two discs?

With few exceptions, the isolation and identification of bacterial pathogens from clinical specimens must be followed by laboratory tests to determine which antibiotics will be most effective in treating the patient. The Kirby-Bauer method for this purpose is based on the diffusion of antibiotics from antibiotic-impregnated paper discs into an inoculated culture medium in a petri plate. The diameter of the area of growth inhibition around the antibiotic disc will give information that is useful for the effective treatment of the patient.

The Kirby-Bauer susceptibility test is performed using a pure culture of previously identified bacterial organism. The technique is carried out with standardized media, inocula, time and temperature of incubation, and antibiotic discs so that results from various laboratories will be comparable and related to the standards associated with the method. The inoculum to be used in this test is prepared by adding growth from 5 isolated colonies grown on a blood agar plate to 5 ml of broth. Several colonies are used to avoid clone differences in susceptibility. This culture is then incubated for 2 hours to produce a bacterial suspension of moderate turbidity. The turbidity is adjusted with sterile medium to match a standard turbidity tube of 1% barium chloride in 1% sulfuric acid (0.36 N). A sterile swab is used to obtain an inoculum from the standardized culture. This inoculum is then streaked evenly in three directions on a Mueller-Hinton plate so that confluent growth of the organism will be obtained.

The antibiotic discs are placed on the surface of the medium at evenly spaced intervals with flamed forceps or a disc applicator. Depending on the organism, different antibiotic-containing discs are used; thus, Gram-positive and Gram-negative organisms are tested against different panels of antibiotics. Antibiotics which produce smaller inhibition zones are best located in the center ring. Incubation is usually overnight with an optimal time being 14 hours. The diameters of the inhibition zones (including the 6 mm disc) are measured using a calipers. A zone size interpretive chart can be used to determine susceptibility or resistance of the organism to each antibiotic. At the University Hospital's Diagnostic Microbiology Laboratory, the caliper is electronically connected to a computer that contains the zone-size interpretive information, and when the technician presses a key, the interpretation appears on the screen as: "susceptible", "intermediate", or "resistant", according to data accumulated from many other tests. Thus, zone sizes are not reported to the clinician but only the three possible interpretations.

In some instances, a physician may wish to have a more quantitative antibiotic susceptibility determination than that obtained using the Kirby-Bauer disc method. This method of testing is called the Minimal Inhibitory Concentration (MIC). Such occasions where more accurate determination of the antibiotic susceptibility of isolated organisms may be required include failure to respond to apparently adequate antibiotic concentrations or relapse while undergoing antibiotic therapy. In these cases, the antibiotic levels in a patient's bloodstream would be monitored to insure that the minimal inhibitory concentration of the antibiotic has been achieved. If not, the dosage could be increased. Other occasions where an MIC would be especially important is for immunosuppressed patients who may not be able to mount an adequate immune response to help fight the infection; or if the antibiotic of choice has the potential to be toxic to the patient when given in a high concentration. By determining the MIC of the antibiotic that would be effective against the organism causing the infection, the dosage could be decreased to reduce the possibility of toxicity.

In the broth tube dilution method for determining the MIC of an organism to a particular antibiotic, specific amounts of the antibiotic are prepared in decreasing concentrations. This dilution series of the antibiotic is then inoculated with a culture of the organism to be tested. The susceptibility of the organism is determined after suitable incubation by macroscopic observation for the presence or absence of growth in the varying concentrations of the antimicrobial agent. This bacteriostatic end-point value is known as the Minimal Inhibitory Concentration (MIC). The Diagnostic Microbiology Laboratory performs MIC tests upon request and for all organisms isolated from normally sterile anatomical sites (e.g., blood, peritoneal fluid, pericardial fluid, spinal fluid, and fluid obtained by suprapubic tap).

A test has been developed that combines the convenience of disk diffusion with the ability to generate MIC data. The E-test (AB Biodisk, Solna, Sweden) utilizes plastic strips that are impregnated on one side with an antimicrobial agent concentration gradient. The other side contains a numeric scale that indicates the drug concentration. Mueller-Hinton plates are inoculated as for the Kirby-Bauer test and the strips are placed on the inoculum lawn. Multiple antimicrobials can be tested on a single isolate. Following overnight incubation, the plate is examined and the number present at the point where the border of growth inhibition intersects the E-strip is taken as the MIC. The same MIC criteria used for dilution methods are used with the E-test value to assign a category of susceptibility or resistance. The E-test is useful for producing MIC data in situations in which the level of resistance can be clinically important (e.g. penicillin or cephalosporins against S. pneumoniae).

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