The genus Neisseria belongs to the family Neisseriaceae.  Other genera in the family are Branhamella, Moraxella, and Acinetobacter.  The Neisseria  genus was named after Dr. Albert Neisser, who discovered the etiological agent for the venereal disease, gonorrhea, in 1879.

 In the clinical laboratory, the nonpathogenic form of these bacteria are found in throat and nasal swabs, urethral exudate, cervical and  vaginal swabs. However,  these specimens may contain the bacteria that cause meningococcal meningitis or gonorrhea whose etiological agents are two pathogenic Neisseriaspecies.  It is important to differentiate between the pathogens from the nonpathogens.  The two important pathogenic Neisseriaare N. meningitidisand N. gonorrhoeae.  These pathogenic species are highly susceptible to drying and exposure to sunlight.  They also autolyze readily and, therefore, are difficult to maintain in the laboratory on culture media.

 Pathogenic Neisseria  species are nutritionally fastidious and microaerophilic, however nonpathogenic are not.  N. meningitidis  and N. gonorrhoeae  require a chocolate blood medium (hemoglobin) and a 3-10% carbon dioxide atmosphere for successful isolation from the specimens.  A popular and current plating medium for their primary isolation is Thayer-Martin chocolate agar which contains certain antimicrobials to make it inhibitory to certain other microorganisms.  Colonies on this selective medium should be tested for oxidase production and cellular morphology.  Gram negative diplococci that are oxidase positive are presumed to be neisseriae.  Confirmatory tests for species identification must be used.

 The oxidase test is a qualitative procedure for determining the presence of cytochrome oxidase activity in bacteria.  The reaction is dependent upon the presence of an intracellular cytochrome oxidase system that catalyzes the oxidation of cytochrome c by molecular oxygen, which then serves as the terminal electron acceptor in the organism’s electron transport system.  N, N, N, N-tetramethyl-p-phenylenediamine dihydrochloride is an aromatic amine which is dimethylated at its two amino groups.  In the reduced state, the reagent is colorless, while in the oxidized state the reagent is dark purple.  In the oxidase test, cytochrome oxidase produced by the microorganism does not directly oxidize the p-phenylenediamine reagent, but rather oxidizes cytochrome c, which in turn oxidizes the reagent to form a purple colored compound.  In essence, the oxidase test determines the presence or absence of cytochrome c.  Organisms containing cytochrome c as part of their respiratory chain are oxidase positive and turn the reagent purple; organisms lacking cytochrome c as part of their respiratory chain do not oxidize the reagent, leaving it colorless within the time limits of the test, and are oxidase negative.
 The type of specimen (primary vehicle) from which the organism was isolated aids in probable identification (e.g., spinal fluid-meningococcus; urethral exudate-gonococcus).  But certain biochemical and/or serological tests should be performed.

 The usually non-pathogenic Neisseria are also found in the respiratory tract as normal inhabitants.  These include N. sicca, N. subflava, N. flavescens. N. mucosa  and N. lactamica.   Moraxella  catarrhalis (formerly Branhamella catarrhalis) is also commonly found in this location.
 

 Below are listed some of the important identifying characteristics of Neisseria species.
 

 cocci (0.6-1.0 um); single and pairs with adjacent sides
 flattened (coffee-bean shaped)

 gram negative

 oxidase (and catalase)  positive

 inhabitants of mucous membranes

 complex growth requirements (for some, especially the pathogens)

 capsules and pili may or may not be present

 optimum temperature is about 37^O C

 some produce pigmented colonies

 (no endospores, no flagella)
 
 

Materials:

  Per pair of students:
 

  1 nutrient agar plate
  1 chocolate blood agar  plate
  1 culture each of N. gonorrhoeae, N. sicca, N. lactamica, and M. catarrhalis
  oxidase reagent
 

Part 1 WEDNESDAY
 

A.  Known cultures: Work as a table group.

 Each table will be given a culture of N. gonorrhoeae (gonococcus), N. sicca, N. lactamica, and Moraxella catarrhalis.

 Each pair of student will carry out the following with each organism:

 1.  Prepare a gram stain and observe.

  NOTE: The tendency of some, especially M. catarrhalis, to resist decolorization.

 2.  Streak for isolation each of the above species by streaking one quadrant of a plate of each media listed below:

  NOTE:  Place very little inoculum on the edge of the quadrant and then use the three sector streak technique.

  NOTE: Media to be used for the growth of gonococcus (or meningococcus) should be warmed to at least to room temperature prior to inoculation.

   a.   nutrient agar plate divided into 4 sectors

   b.   chocolate blood agar plate divided into 4 sectors. This medium is nonselective.

   NOTE:  The selective version of the chocolate blood agar is called Thayer-Martin and contains antimicrobial compounds which inhibit the growth of contaminants  from original specimens. Several variations of this original medium are available. These may contain vancomycin, sodium colistimethate, trimethoprim, and nystatin, along with a defined supplement added to a chocolate agar base medium, which gives superior recovery of N. gonorrhoeae from genital infection specimens.  The antimicrobial content is sufficient to inhibit several normal flora neisseriae and certain other gram positive and gram negative bacteria, as well as some  yeasts.

 3.   Incubate plates at 37° C in a humidified candle jar (insertion of a piece of
       dampened paper towel is satisfactory for providing a humid atmosphere).

 4.   Examine at 24 and 48 hours.

 5.  Perform the oxidase test .  Apply 1-2 drops of fresh oxidase reagent    (tetramethyl-p-phenylenediamine dihydrochloride) to the colony of each stock  culture and record results.

  NOTE:  A positive oxidase test is given by a darkening of the colony, eventually turning purple.   Reactions are positive for Moxaxella catarrhalis, and the Neisseria; however, other organisms such as Alkaligenes,  Pseudomonas, and certain other bacteria and yeasts sometimes found in the normal throat, cervix, urethra, and other body areas may also give a positive test.  Nevertheless, the test is useful when coupled with morphological (and biochemical) tests for the identification of Neisseria.

  NOTE:  The application of the oxidase reagent to the colonies will kill the organisms within 15 minutes. Therefore, if subcultures are to be made, they should be completed within this time.
 
 

Part 2  WEDNESDAY

A.  Throat Isolates:  Work individually.

 1.   A nasopharyngeal swab from yourself should be spread over a relatively small area (2-3 cm2) of a chocolate blood agar plate. This inoculum is then streaked to obtain isolated colonies.

 2.   Incubate the plate under microaerophilic conditions (candle jar) at 37^O C.

 3.   Examine plates at 24 or 48 hours.
 
 
 

Part 3  THURSDAY

A.   Known cultures:

   1.   Describe colonial characteristics on chocolate and nutrient agar plates.

 NOTE:  the characteristics of typical colonies of N. gonorrhoeae, N. sicca,
  N. lactamica, and M. catarrhalis, if any, on nutrient and chocolate blood agar.

  3.   Tabulate results-discuss colonial features and ability to grow on nutrient agar.

B.    Isolates from throat cultures:

  1.   Examine the colonies on the plate with oblique, reflected light.

  2.   Test colonies suspected of being neisseriae with a small drop of the oxidase reagent.

  3.  Prepare gram stains of oxidase-positive colonies.

 NOTE:  You will not need to prepare subcultures of the isolates.

 3.  Record your results

 4.  Discuss the results in light of numbers.
 
 

DEMONSTRATION

 1.  Observe the gram stain of a urethral exudate smear from a gonorrhea patient.

 2.  Describe  your observations.