Microbiology Laboratory Turkey

Mikrobiyoloji Ile Ilgili Tüm Konuların Kısa ve Öz Anlatımları. Microbiology Lab Information.

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coagulase test etiketine sahip kayıtlar gösteriliyor. Tüm kayıtları göster
coagulase test etiketine sahip kayıtlar gösteriliyor. Tüm kayıtları göster

24 Ekim 2018 Çarşamba

Ekim 24, 2018

STAPHYLOCOCCUS SPP. (ENGLISH)

STAPHYLOCOCCUS SPP.

Staphylococcus bacteria are pathogens to both man and other mammals. They are gram positive bacteria that are small round in shape (cocci) and occur as clusters appearing like a bunch of grapes on electron microscopy.

Coagulase Reaction
Staphylococcus were earlier divided into two groups on the basis of their ability to clot blood plasma. The coagulase-positive staphylococci constitute the most pathogenic species S aureus. The coagulase-negative staphylococci (CNS) are now known to comprise over 30 other species. It is the CNS that is present as harmless bacteria on skin but some of these may cause infections as well.
These days coagulase reaction is no longer used to classify S.aureus. This is because coagulase is a marker for S aureus but there is no direct evidence that it is a virulence factor. Nevertheless, the term is still in widespread use among clinical microbiologists.
Proteins and Virulence
S. aureus expresses certain proteins and polysaccharides on its surface. This is correlated with virulence. Virulence is the effect of many factors expressed during infection. The bacteria also produce certain toxins. In the body antibodies neutralize staphylococcal toxins and enzymes.

Taxonomy and Naming Conventions
At least 30 species of staphylococci have been recognized by biochemical analysis. This is especially so with DNA-DNA hybridization. Of these, 11 are found in humans as harmless bacteria on skin, in the nose and throat. These can cause disease and infections in certain situations.

Identifications Under The Microscope
These bacteria are Gram positive (+) cocci about 0.5 – 1.0 μm in diameter. They are present as grape like clusters. They may also occur in pairs and occasionally in short chains. The clusters arise because staphylococci divide in two planes. This clustering helps to distinguish staphylococci from streptococci, which usually grow in chains.
Growing on solid medium colonies of S. aureus these appear as golden clumps.

Catalase Test
This test helps to distinguish between streptococci (that is catalase-negative) and staphylococci (which are catalase positive). On an agar slant or broth culture of the bacteria several drops of 3% hydrogen peroxide are applied. Catalase-positive cultures bubble at once. This cannot be done on blood agar since blood itself will produce bubbles.

Identification of Staphylococcus aureus
After sample from the lesions are taken, they can be stained with Gram stain. S. Aureus is Gram positive. The organism from the clinical specimen from blood culture or pus is then streaked over solid media such as blood agar, tryptic soy agar or heart infusion agar. If the specimen is suspected to be contaminated it is plated on mannitol salt agar containing 7.5% sodium chloride.

Another test is production of thermostable deoxyribonuclease. S aureus can be confirmed by testing colonies for agglutination with latex particles coated with immunoglobulin G and fibrinogen which bind protein A and the clumping factor, respectively.

Microscopically cells occur singly and in pairs, short chains, and grape-like clusters. The cell wall of the bacteria contains teichoic acid. Ribitol teichoic acid (Polysaccharide A) is present in Staphylococcus aureus. Protein A uniformly coats surface of S. aureus and is usually oxidase negative.

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3 Ekim 2018 Çarşamba

Ekim 03, 2018

BASİT BOYAMA

BASİT BOYAMA

Mikroorganizmaların ve özellikle bakterilerin mikroskobik morfolojilerinin incelenebildiği boyama yöntemidir. Bu amaçla farklı boyalar kullanılsa da en yaygını Löffler'in metilen mavisi çözeltisidir.
Bakteriler, kullanılan boyanın rengini alır.


Basit boyama yönteminde aşağıdaki işlemler sırasıyla uygulanır:

➧ 1.2’de belirtildiği şekilde preparat hazırlanır (yayma, kurutma, tespit). 
➧ Preparatın üzerine boya çözeltisi eklenerek kaplanır. 
➧ Yeterli süre (Löffler'in metilen mavisi kullanılmışsa 30–60 saniye, karbol fuksin kullanılmışsa 5–10 saniye, kristal viyole kullanılmışsa 20–30 saniye) beklenir. Eğer daha uzun süre temas olursa bakteriler ile diğer cisimlerin ayırt edilmesi güçleşir. 
➧ Boya dökülerek preparat distile suyla yıkanır. 
➧ Preparat havada veya kurutma kâğıdı ile kurutulur. 
➧ Mikroskopta incelenir. 


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2 Ekim 2018 Salı

Ekim 02, 2018

COAGULASE TEST

COAGULASE TEST


Coagulase test is done to distinguish Staphylococcus aureus from other Coagulase negative Staphylococcus (CONS) like Staphylococcus epidermidis, Staphylococcus saprophyticus etc. Staphylococcus  aureus produces two types of coagulase, i.e. free coagulase and bound coagulase. Free coagulase is an extracellular enzyme that can be detected in tube coagulase test while bound coagulase is a cell wall associated protein that can be detected in slide coagulase test.



Principle of Tube Coagulase Test:

The free coagulase secreted by S. aureusreacts with coagulase reacting factor (CRF) present in plasma to form a complex, thrombin. This converts fibrinogen to fibrin resulting in clotting of plasma.

Procedure For Tube Coagulase Test:

  1. Take three test tubes and label them as “test”, “negative control” and “positive control”.
  2. Fill each test tube with 1 ml of 1:6 dilution of rabbit or human plasma in normal saline.
  3. Add 0.1 ml of overnight broth culture to the tube labeled test. Also add 0.1 ml of overnight broth culture of known S. aureus to the tube labeled positive control and 0.1 ml of sterile broth to the tube labeled negative control.
  4. Incubate all the tubes at 37oC and observe up to four hours.

                                                                    
                                           


Principle of Slide Coagulase Test:


Bound coagulase is also known as clumping factor. It cross-links α and β chain of fibrinogen in plasma to form fibrin clot that gets deposited on the cell wall of the cocci. As a result, individual coccus sticks to each other and clumping can be observed.

Procedure for Slide Coagulase Test:
  1. Emulsify few colonies of Staphylococci from culture in a drop of normal saline on two ends of clean glass slide.
  2. Label one as “test” and the other as “control”. The control suspension serves to rule out false positives due to autoagglutination.
  3. Mix a drop of rabbit or human plasma with the test suspension.
  4. Observe agglutination or clumping of cocci. Agglutination within 5-10 seconds is considered as positive. Some strains of S. aureus may not produce bound coagulase, and such strains  must be identified by tube coagulase test.



Positive result is indicated by clotting of the plasma, which remains in place even after inverting the tube. If the test remains negative until four hours at 37oC, leave the tube at room temperature for overnight incubation. Some strains on continued incubation produce fibrinolysin that lyses the clot.
Slide coagulase test is useful as a screening test while tube coagulase test is useful in confirmation of coagulase test . Not all strains of S. aureus produce coagulase, such rare strains are identified by thermonuclease test. Some species of Staphylococci such as S. lugdunensis and S. schleiferi  are slide coagulase positive while S. hyicus and S. intermedius are tube coagulase positive.
Note: Citrated plasma should not be used as some citrate utilizing bacteria (Pseudomonas) may utilize citrate to give false positive result.


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Ekim 02, 2018

KOAGÜLAZ TESTİ

KOAGÜLAZ TESTİ


 Koagülaz, özellikle patojen Staphylococcuslar tarafından üretilen ve fibrinojeni fibrine dönüştürerek plazmayı pıhtılaştıran bir enzimdir. Koagülaz testi, Staphylococcus aureus’un diğer Staphylococcuslardan ayırt edilmesinde kullanılan en önemli, enzimatik bir testtir. Staphylococcuslarda; serbest koagülaz ve bağlı koagülaz olarak iki enzim türü vardır. Serbest koagülazın varlığı tüpte koagülaz testiyle, bağlı koagülazın varlığı ise lamda koagülaz testiyle tespit edilir. Tüpte koagülaz testi, lamda koagülaz testine göre daha güvenilir sonuç verdiğinden tercih edilir. 

Testlerde reaktif olarak taze hazırlanmış ya da ticari olarak dondurularak kurutulmuş tavşan plazması kullanılır. Tavşan plazması, bazı patojenik bakteri türleri tarafından sentezlenen ve koagülaz enzimi tarafından fibrine dönüştürülen fibrinojeni içerir.




➤ Tüpte Koagülaz Testi 

Bu yöntemde amaç, bakterilerin bulundukları ortama saldıkları “bağlı olmayan-serbest koagülaz” enzimini tespit etmektir. 

Testin yapılış tekniği; 

↣ Staphylococcus şüphesi olan kolonilerden 0,5 ml alınıp tüpteki tavşan plazmasına eklenir. 
↣ Etüvde, 37oC’de 1- 4 saat süreyle bekletilir. 
↣ Plazmanın pıhtılaşıp akışkanlığını kaybetmesi yani, belirgin pıhtı oluşması pozitif, akışkanlığını sürdürmesi ise negatif olarak değerlendirilir. Pıhtı oluşmaması halinde süspansiyon oda ısısında 24 saate kadar bekletilir. Çünkü nadiren de olsa bazı izolatlar, geç dönemde plazmanın pıhtılaşmasına yol açabilir. 



➤ Lamda Koagülaz Testi 

Bu yöntemde amaç, bakterinin yüzeyinde bulunan bağlı koagülazı tespit etmektir. Testin yapılış tekniği; 

⇨ Lam üzerine bir damla distile su konur. 
⇨ Üzerine özeyle şüpheli bakteri kolonisinden alınıp karıştırılır. 
⇨ Daha sonra bir öze dolusu tavşan plazması ilave edilerek karıştırılır. 
⇨ 1-2 dakika içinde kümeleşme-pıhtılaşma olup olmadığına bakılır. 
⇨ Pıhtılaşma oluşması pozitif, oluşmaması ise negatif olarak değerlendirilir.


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