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.Neisseria Lab

الكلية كلية طب الاسنان     القسم  العلوم الاساسية     المرحلة 3
أستاذ المادة زينب خضر احمد المهدي الامين       18/03/2020 13:30:51
Neisseria
Is a large genus of bacteria that colonize the mucosal surfaces of many animals. Of the 11 species that colonize humans, only two are pathogens, N. meningitidis and N. gonorrhoeae.

Microscopy
N. meningitidis are gram-negative, coffee-bean shaped diplococci that may occur intracellularly or extracellularly in PMN leukocytes. N. meningitidis is a fastidious organism, non motile, non spore forming, nonencapsulated, which grows best at 35-37°C with ~5% CO (or in a candle-jar). It can grow on both a blood agar plate (BAP) and a chocolate agar plate (CAP). Colonies of N. meningitidis are grey and unpigmented on a BAP and appear round, smooth, moist, glistening, and convex, with a clearly defined edge. N. meningitidis appear as large, colorless-to-grey, opaque colonies on a CAP. Prior to identification and characterization testing procedures, isolates should always be inspected for purity of growth and a single colony should be re-streaked, when necessary, to obtain a pure culture. For the following identification and characterization procedures, testing should be performed on 18-24 hour growth from a BAP (Figure 1) or a CAP (Figure 2) at 35-37°C with ~5% CO (or in a candle-jar).
Clinical samples:

Blood and/or spinal fluid for (N. meningitidis ), vaginal or penile discharge (for N. gonorrhoeae)

Identification

The following tests are recommended to confirm the identity of cultures that morphologically appear to be N. meningitidis (Figure 3). N. meningitidis can be identified using Kovac’s oxidase test and carbohydrate utilization. If the oxidase test is positive, carbohydrate utilization testing should be performed. If the carbohydrate utilization test indicates that the isolate may be N. meningitidis, serological tests to identify the serogroup should be performed. This sequence of testing is an efficient way to save costly antisera and time. Additional methods for identification and characterization of N. meningitidis using molecular tools are PCR methods and other molecular methods.
Kovac’s oxidase test

Kovac’s oxidase test determines the presence of cytochrome oxidase. Kovac’s oxidase reagent, tetramethyl-p-phenylenediamine dihydrochloride, is turned into a purple compound by organisms containing cytochrome c as part of their respiratory chain. This test aids in the recognition of N. meningitidis, but other members of the genus Neisseria, as well as unrelated bacterial species, may also give a positive reaction. Positive and negative quality control (QC) strains should be tested along with the unknown isolates to ensure that the oxidase reagent is working properly.

Performing Kovac’s oxidase test Filter paper method
1. Grow the isolate (s) to be tested for 18-24 hours on a BAP at 35-37°C with ~5% CO (or in a candle-jar).
2. On a nonporous surface (i.e., Petri dish or glass plate), wet a strip of filter paper with a few drops of Kovac’s oxidase reagent.
3. Let the filter paper strip air dry before use.
4. Use a disposable plastic loop, a platinum inoculating loop, or a wooden applicator stick to pick a portion of a colony from overnight growth on the BAP and rub it onto the treated filter paper (Figure 4).
5. Observe the filter paper for color change to purple.
6. Perform steps 3 and 4 with a positive and negative QC strain to ensure that the oxidase reagent is working properly.
C. Reading the oxidase test results

Positive reactions will develop within 10 seconds in the form of a purple color where the bacteria were applied to the treated filter paper. Delayed reactions are unlikely with N.
meningitidis.

• Negative reactions will not produce a color change on the treated filter paper.
Neisseria gonorrhoeae
Neisseria gonorrhoeae, also known as gonococci (plural), or gonococcus (singular), is a species of Gram-negative coffee bean-shaped diplococci bacteria responsible for the sexually transmitted infection gonorrhea.
Neisseria are fastidious Gram-negative cocci that require nutrient supplementation to grow in laboratory cultures. To be specific, they grow on chocolate agar with carbon dioxide. These cocci are facultatively intracellular and typically appear in pairs (diplococci), in the shape of coffee beans. Of the eleven species of Neisseria that colonize humans, only two are pathogens. N. gonorrhoeae is the causative agent of gonorrhea and is transmitted via sexual contact.
Culture and Isolation
Neisseria is usually isolated on Thayer-Martin agar (or VPN agar)—an agar plate containing antibiotics (vancomycin, colistin, nystatin, and trimethoprim) and nutrients that facilitate thegrowth of Neisseria species while inhibiting the growth of contaminating bacteria and fungi. Further testing to differentiate the species includes testing for oxidase (all clinically relevant Neisseria show a positive reaction) and the carbohydrates maltose, sucrose, and glucose test in which N. gonorrhoeae will oxidize (that is, utilize) only the glucose.
References
Ryan KJ; Ray CG (editors) (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. ISBN 0-8385-8529-9.


A.Prof. Dr. Zainab Al-Mahdi


المادة المعروضة اعلاه هي مدخل الى المحاضرة المرفوعة بواسطة استاذ(ة) المادة . وقد تبدو لك غير متكاملة . حيث يضع استاذ المادة في بعض الاحيان فقط الجزء الاول من المحاضرة من اجل الاطلاع على ما ستقوم بتحميله لاحقا . في نظام التعليم الالكتروني نوفر هذه الخدمة لكي نبقيك على اطلاع حول محتوى الملف الذي ستقوم بتحميله .