EXERCISE 11

GRAM-NEGATIVE PYOGENS

I. INTRODUCTION

The pyogenic organisms studied in this exercise are primarily pathogens of the upper respiratory tract although they may be found in the absence of disease in the nasopharynx. The important genera are Neisseria and Hemophilus.

NEISSERIAE

The Neisseriae comprise a family of Gram-negative organisms most of which are non-pathogenic inhabitants of the upper respiratory tract. They are cocci that occur in pairs with adjacent surfaces slightly flattened to give them a bean-shaped appearance. All of the species in this family produce the enzyme oxidase, and so can be differentiated from most other Gram-negative bacteria (except Pseudomonas) by the Oxidase Test.

Two members of this family are important pathogens with humans being the sole reservoir of these two species. Neisseria meningitidis causes purulent meningitis, occasionally epidemic in distribution. Infections due to Neisseria gonorrhoeae have presently reached epidemic proportions. Primarily a venereal infection, sequelae to initial infection may consist of septic arthritis and endocarditis.

Branhamella sicca, Branhamella flavescens and other members of the Neisseriae family are normal flora of the nasopharynx, and can also be found in the female genital tract. These are usually non-pathogenic and thus must be differentiated from the pathogens in cultures taken from areas contaminated with normal flora. Although placed in a different genus (Branhamella), these organisms exhibit gram stain reactions identical to the pathogenic Neisseria species.

HEMOPHILUS

The Hemophilus group are Gram-negative, non-motile, aerobic, pleomorphic bacilli. These organisms require enriched media for primary isolation and all grow better in the presence of blood (Hemophilus = blood loving). The factors essential for the growth of some or all of the species in this genus are a heat-stable factor (X- factor) identified as hemin or hematin, and a heat-labile factor (V-factor) replaceable by nicotinamide adenine dinucleotide (NAD) or closely related compounds.

II. LAB WORK

• 2 Blood Agar Plates (BAP) with unknown Neisseriae cultures (pink dot)
• 2 Chocolate Agar Plates (CAP) with unknown Hemophilus cultures (blue dot)
• 2 Nutrient Agar plates (NA) each with a Hemophilus culture and X-V factor strips
• 1 Oxidase reagent dropper vial
• 2 racks of Carbohydrate Utilization tubes, inoculated with unknown Neisseriae

1. Each pair of students will prepare a Gram-stain of one of the Neisseriae and one of the Hemophilus cultures.
2. Perform the Oxidase test on each of the Neisseriae cultures:
   1. Carefully break the glass ampule inside the Oxidase reagent vial
   2. Place one drop of reagent (do no remove cap; it has a dropper hole) on several isolated Neisseriae colonies.
   3. Observe that these colonies rapidly change color to black. This is a positive reaction indicating the presence of the enzyme, oxidase.
3. Observe the material on the front bench that demonstrates the ability of non-pathogenic Neisseriae to grow at room temperature (22°C), and use the table below and your Carbohydrate Utilization tubes to identify your Neisseriae unknowns.
4. Fill in your Report Sheet and hand it in at the end of lab TODAY.

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   SPECIES	Growth on Nutrient Agar @ 22°C	CARBOHYDRATE UTILIZATION
   		GLUCOSE (red)	MALTOSE (green>	SUCROSE (black)
   N. gonorrhoeae	-	+	-	-
   B. flavescens	+	-	-	-
   N. meningitidis	-	+	+	-
   B. sicca	+	+	+	+

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5. Differentiation of Hemophilus species
   1. Observe the 2 TSA plates containing X-V factor rings.
   2. Note that an Hemophilus culture will only grow where all of its nutritional requirements are being met.
   3. Check the table below and use the X and/or V factor requirements for growth to identify the 2 Hemophilus unknown cultures.

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   SPECIES	GROWTH FACTOR REQUIREMENTS		HEMOLYSIS
   	Hemin (X factor)	NAD (V factor)
   H. influenzae	+	+	-
   H. parainfluenzae	-	+	-

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   1. Fill in your Report Sheet and hand it in at the end of lab TODAY.