Hemolysis Types Blood Agar

Susceptibility to optochin (ethylhydrocupreine hydrochloride) is a simple and reliable method of differentiating Streptococcus pneumoniae from other alpha-haemolytic Streptococci.


TEST PRINCIPLE
The optochin test detects an organisms susceptibility to the chemical optochin ethylhydrocupreine hydrochloride). The chemical tests the fragility of the bacterial cell membrane and causes Streptococcus pneumoniae to lyse due to changes in surface tension.
The optochin test is widely used in the form of filter paper discs, impregnated with ethylhydrocupreine hydrochloride which are applied directly to inoculated plates before incubation.
REAGENTS AND EQUIPMENT
Filter paper discs impregnated with 5µg of ethylhydrocupreine hydrochloride
Bacteriological straight wire/loop (preferably nichrome) or disposable alternative

Quality Control Organisms
Positive control: Streptococcus pneumoniae 
Negative control: Streptococcus viridans 


METHOD/PROCEDURE AND RESULTS
Pure colony

• Streak a blood plate with the organism to be tested
• Place an optochin disc in the centre of the inoculum
• Incubate at 35-37o C for 18-24h in 5% CO2
• Examine for zones of inhibition 

Basitrasin testinde disk etrafında zon oluşumu

A bacterium is a single-celled (unicellular) microorganism that does not have a nucleus or any other membrane-bound organelles. Bacteria are sometimes called 'prokaryotes.' In Greek, 'prokaryote' literally means 'before the nut' (where 'the nut' is the nucleus.)

Bacteria adapt to become well-suited to their environments, and therefore come in many shapes and forms. However, they all have a few parts in common.

BACTERIA

Classification
Because bacteria are so diverse in both form and habitat, biologists have struggled with their classification, (also called their 'taxonomy.') For many years, bacteria were called 'monerans,' and placed in a kingdom of the same name. Although you still might hear bacteria referred to as monerans, it's not a term currently accepted among biologists.
Life is now classified into three domains; a domain is a taxonomic level that is higher than a kingdom, and based on an organism's DNA. Bacteria is one of these domains, while the others are eukarya, composed of organisms that have nucleated cells, and archaea, composed of unicellular prokaryotes, many of which have evolved to exploit extreme environments. Organisms in the archaea domain were originally considered a type of bacteria - known as archaebacteria - but scientists have since reclassified them. In this image, groups of archaea are indicated by green lines, and bacteria are in blue. Red lines indicate eukaryotes.



How we group bacteria
Scientists try to make sense of the sheer number and diversity of bacteria by grouping them in various ways. Here are just a few ways that are accepted in the scientific and medical communities:
Shape: Bacteria are usually one of three shapes. Cocci are round; bacilli are rod shaped; spirilla (also called spirochetes) are spiral.

Staphylococcuslar hareketsiz, sporsuz, genellikle kapsülsüz, gram pozitif (+) bakterilerdir. Mikroskobik incelemede; yuvarlak koklardan oluşmuş Staphylococcus hücreleri, üreme sırasında birbirinden ayrılmayıp 3 boyut yönünde çoğaldıklarından üzüm salkımına benzer kümeler halinde görünür. 

Üreme Özellikleri 
Staphylococcuslar fakültatif anaerobdur. Optimal üreme ısıları 37oC, optimal pH'ları 7.27.4'tür. Kuruluğa, ısıya, çeşitli antiseptik ve dezenfektanlara birçok bakteriye göre daha  dayanıklıdır; ancak kristal viyole ve malaşit yeşili gibi boyalara hassastır. Adi besiyerinde kolay ürer. Bu besiyerlerine kan ve glikoz ilavesiyle bakterinin üremesi artar. Bu tür ortamlarda S tipi koloni oluşturur. Koloniler, üreme ortamında renkleri ile ayrıcalık gösterir ve bazı Staphylococcusların oluşturduğu pigmentin rengini alır. 

➤ İnsanlarda enfeksiyona neden olan başlıca Staphylococcus türleri şunlardır; 
➧ Staphylococcus aureus, en çok enfeksiyona neden olan patojen türüdür. 
➧ Staphylococcus epidermidis, üst solunum yolu ve derinin florasında bulunur; vücut direnci düştüğünde fırsatçı enfeksiyonlara neden olur. 
➧ Staphylococcus saprophyticus, normal florada bulunmaz. Vücut direnci düştüğünde başta üriner sistem enfeksiyonları olmak üzere fırsatçı enfeksiyonlara neden olur. 
Antijenik Özellikleri 
Staphylococcuslar, antijen yapıları bakımdan kesin bir özellik göstermez. Staphylococcus aureus'un tiplendirilmesinde faj tiplendirmesi kullanılır. Gerektiğinde serolojik tiplendirmelerden de yararlanılır. 
Staphylococcus aureus’un Toksin ve Enzimleri 

• Hemolysin; eritrositleri hemoliz eden toksindir. 
• Lökosidin; lökositleri öldürücü etki gösteren, fagositozu önleyen toksindir. 
• Nekrotoksin; dokuları öldürücü etki gösteren toksindir. 
• Fibronilizin; fibrini eritip bakteriyi hapsolmaktan kurtaran toksindir. 
• Enterotoksin; besin zehirlenmesine neden olan toksindir 
• Plazma koagülaz enzimi; pıhtılaşması önlenmiş kanın plazmasını pıhtılaştıran ve patojenliği gösteren bir enzimdir. 
• Hyalüronidaz enzimi (yayılma faktörü); bakterinin doku içinde yayılmasını sağlayan enzimdir. 
• Penisilinaz enzimi; penisilinin yapısını bozarak etkisini ortadan kaldıran enzimdir. 
• Jelatinaz enzimi; jelatini eriten enzimdir. 

Yaptığı Hastalıklar 
➨Deri Enfeksiyonları; apse, fronkül (çıban), sivilce, impetigo (iltihaplı deri enfeksiyonu), kan çıbanı, hordeolum (arpacık), paronişya (dolama), yara, yanık ve cerrahi yara enfeksiyonları.  
➨Solunum Yolu Enfeksiyonları; anjin, akut veya kronik farenjit, sinüzit, otitis media, stafilokok pnömonisi. 
➨Ürogenital Sistem Hastalıkları; piyelonefrit, salpenjit, otitis media, endometrit, servisit, böbrek apseleri. 
➨Sinir Sistemi Hastalıkları; menenjit, artrit, osteomyelit, besin zehirlenmesi ve sepsis yapar. 

Laboratuvar Teşhisi  
İnceleme örnekleri: Balgam, cerahat, BOS, kan . 
Teşhis yöntemleri 
➧Mikrobiyolojik örneğin direkt mikroskobik incelemesi; direkt preparat hazırlanıp gram boyasıyla boyanarak mikroskopta incelenir. 
➧Kültür; kanlı agara ekim yapılarak üreyen bakterinin koloni ve hemoliz özellikleri incelenir. İzole edilen bir Staphylococcusun Staphylococcus aureus olup olmadığı; jelatinli besiyerine ve chapman besiyerine ekim yapılarak belirlenir. 
➧Biyokimyasal testler; katalaz ve koagülaz testleri yapılır. 

The Three Types of Hemolytic Activities:
➤ a clear zone around bacterial growth -RBC hemolyzed completely (Beta-hemolysis and pathogenic)
➤ a greenish zone around growth -RBC partially hemolyzed (Alpha-hemolysis)
➤ no change around growth -blood is not hemolyzed (Gamma-hemolysis or no hemolysis)



Procedure for Testing an Unknown:
➧ The BAP test may be used to either test a throat culture or an unknown bacterial species for hemolytic activity.
➧ If an unknown species of bacteria is to be tested, divide a BAP plate in thirds.
➧ Inoculate one third with the unknown, one third with a negative control (such as E. coli), and one third with a positve control (such as Bacillus cereus).
➧ It is best to streak your bacteria in a long W pattern using your inoculating needle with plenty of space between the streak lines.  This will put some space between the streaks making it easier to observe the results.  If there is too much growth, which may happen with a swab, waste products may accumulate which may lyse the blood giving the appearance of hemolytic toxins when they are not actually present.
➧ If a candle jar is not available, take a needle and stab a region of heavy inoculation to inoculate bacteria under the agar surface.  This allows for growth of the bacteria under reduced oxygen.  If a candle jar is available (or some other means to reduce the oxygen), do not stab the plates and incubate them in a candle jar.  The cultures are put in the jar, a lit candle is added, the lid is put on tightly, the candle uses up most of the oxygen and goes out, and then the jar is incubated under reduced oxygen.).  Some hemolysins are oxygen sensitive, so the bacteria should be grown and tested under reduced oxygen.  Both the stab and the candle jar reduce the oxygen allowing for the the testing of oxygen sensitive hemolysins.  Oxygen stable hemolysins are not affected by either treatment.
➧ Incubate the plates for 24 hours at 35 degrees. 



Procedure for Testing a Throat Culture:
⇒ If using a throat culture, obtain a swab of the throat and inoculate it in a small area on a BAP plate.
⇒ It is not required but is a good idea to streak the rest of the plate with a loop to try to isolate colonies from the throat culture.  Alternatively, one may swab the entire plate with the throat culture.
⇒ If a candle jar is not available, take a needle and stab a region of heavy inoculation to allow anaerobic growth of the bacteria or at least growth with reduced oxygen.  Otherwise, do not bother with the stabbing and incubate appropriately labeled plates in a candle jar.  (See above.)
⇒ Incubate the plates for 24 hours at 35 degrees.


Results:
Observe your plates for hemolytic activity and record your results as alpha, beta, or gamma (α, β, γ).  Holding the plate up to a light source helps.

Instructor Notes:
Different types of blood (sheep versus horse, etc.) can affect the hemolytic activity of strains.  Optimal hemolytic activity occurs under reduced or anaerobic conditions, but this is often not required.  Hemolytic activity is much easier for students to read and interpret with the use of a candle jar compared to stabbing into the agar, so this method is recommended.  Over incubation of the plates may cause overgrowth which may cause waste products to accumulate which may lyse the blood giving the appearance of hemolytic toxins when they are not actually present.  Many enterics give false hemolytic activity when overgrown.  The plates can be refrigerated if needed to prevent over incubation.