Hayvanların bağırsağında bulunan Escherichia coli’nin tanısında, biyokimyasal idendifikasyon için saf kültürden Eijkman testi yapılır. Bakterinin tanımlanmasında Eijkman testinden daha çok İMVİC testi yapılır; sonuç (+ + - -) şeklinde çıkar yani Escherichia coli’de, indol testi (+), Metilen kırmızısı testi (+), Voges-Proskauer agar testi (-), sitrat testi (-)’dir.

Diagnosis
Salmonellosis should be considered in any acute diarrheal or febrile illness without obvious cause. The diagnosis is confirmed by isolating the organisms from clinical specimens (stool or blood).

Diagnosis
The diagnosis of salmonellosis requires bacteriologic isolation of the organisms from appropriate clinical specimens. Laboratory identification of the genus Salmonella is done by biochemical tests; the serologic type is confirmed by serologic testing. Feces, blood, or other specimens should be plated on several nonselective and selective agar media (blood, MacConkey, eosin-methylene blue, bismuth sulfite, Salmonella-Shigella, and brilliant green agars) as well as intoenrichment broth such as selenite or tetrathionate. Any growth in enrichment broth is subsequently subcultured onto the various agars. The biochemical reactions of suspicious colonies are then determined on triple sugar iron agar and lysine-iron agar, and a presumptive identification is made. Biochemical identification of salmonellae has been simplified by systems that permit the rapid testing of 10–20 different biochemical parameters simultaneously. The presumptive biochemical identification of Salmonella then can be confirmed by antigenic analysis of O and H antigens using polyvalent and specific antisera. Fortunately, approximately 95% of all clinical isolates can be identified with the available group A-E typing antisera. Salmonella isolates then should be sent to a central or reference laboratory for more comprehensive serologic testing and confirmation.

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Morfolojisi 
 Klebsiella pneumoniae hareketsiz, sporsuz, geniş kapsüllü, gram negatif  (-) basildir. Genellikle uç uca, ikişer ikişer kısa zincirler halinde ve tek tek bulunur. Bakteriyolojik boyalarla iyi boyanır.

Üreme Özellikleri 
Klebsiella pneumoniae, aerob ve fakültatif anaerobdur. Optimal üreme ısısı 37oC, optimal pH‟sı 7.4‟tür. Kuruluğa dirençli olup özellikle organik maddeler içinde kurutulurlarsa aylarca canlı kalır. Isıya dayanıksız olup nemli ısıda 55 oC‟de yarım saate ölür. Sıvı besiyerinde homojen bir bulanıklık, yüzeyde zar ve dipte mukoid bir çöküntü oluşturarak ürer. Katı besiyerinde mukuslu ve kapsüllü olduğu için öze ile alındığında sünen M tipi koloniler oluşturur; kanlı jelözde hemoliz oluşturmaz. İndol, H2S, Voges-Proskauer, metil kırmızısı testleri negatif, üre ve KCN testleri pozitif olan bakterilerdir.

Yaptığı Hastalıklar 
 Klebsiella pneumoniae; bulunduğu yerlerde uygun koşulların oluşması veya yer değiştirerek diğer organ ve sistemlere yerlemeleri halinde, başta pnömoni olmak üzere, anjin, sinüzit, otitis media, üst solunum yolu enfeksiyonu, gastroenterit, septesemi, menenjit, kolesistit ve üriner sistem hastalıklarına neden olur.

 Laboratuvar Teşhisi  
➨ İnceleme örnekleri: İdrar, balgam, kan, dışkı, boğaz sürüntüsü, cerahat. 
➨Teşhis yöntemleri 
➧ Mikrobiyolojik örneğin direkt mikroskobik incelemesi; santrifüj edilen idrardan hazırlanan preparatın incelenmesinde; bol lökosit ve ikişerli hareketsiz bakterilerin görülmesiyle ve hazırlanan preparatın gram boyasıyla boyanarak incelenmesiyle de teşhis edilir.
➧ Kültür; EMB AGAR, Endo, Mac-Conkey Agar besiyerine ekim yapılır.
➧ Biyokimyasal Testler; tipik mukoid kolonilerden çeşitli biyokimyasal testlerle teşhis edilir.
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Amaç, B grubu Streptococcusların oluşturduğu CAMP (Christie, Atkins, Munch-Peterson) faktörünü tespit etmektir. Bu testi bulan bilim adamlarının ilk harfleri alınarak CAMP adı oluşturulmuştur.



Testin yapılış Tekniği;

➤ Bazı Staphylococcusların (Staphylococcus aureus) var olduğu düşünülen örnekten,
özeyle alınarak %5‟lik koyun kanlı agarın ortasına boydan boya kalın bir çizgi
şeklinde ekilir.
➤ Bu ekim çizgilerine dik doğrultuda, Staphylococcus ekim çizgisine dokundurmamaya
dikkat ederek incelenecek mikroorganizmadan 2-3cm uzunlukta ekim yapılır.
➤ Plaklar etüvde, 37°C'de 18-24 saat inkübe edilir.
➤ İnkübasyon sonunda, Streptococcus ve Staphylococcusların üreme yapıları incelenir.
Birbirlerine yakın oldukları yerde ok başı şeklinde hemoliz meydana gelirse
CAMP testi pozitif olarak değerlendirilir.

Objective

To determine the ability of the organism to hydrolyse urea by the action of urease enzyme.

Principle

Urea is a nitrogen-containing compound that is produced during decarboxylation of the amino acid arginine in the urea cycle. Urea is a major organic waste product of protein digestion in most vertebrate and is excreted in urine. Some bacteria have the ability to produce an enzyme urease as part of its metabolism to break down urea. The urease is a hydrolytic enzyme which attacks the carbon and nitrogen bond with the liberation of ammonia and carbon dioxide. It is a useful diagnostic test for identifying bacteria, especially to distinguish members of the genus Proteus from Gram-negative pathogens. Proteus vulgaris is an important and fast producer of urease.

Urease test is performed by growing test organism on urea agar slant or urea broth with phenol red as an indicator with pH6.8. During the incubation period, the organism capable of producing urease enzyme hydrolyse urea and produce ammonia that raises the pH level. As the pH increases, the phenol red changes from yellowish orange as the initial colour of medium to deep pink. Failure of development of a pink colouration due to no ammonia production is evidence of an inability of the organism to produce urease enzyme.

Media

Enzymatic digest of gelatin (1 g), dextrose (1 g), NaCl (5 g), KH2PO4 (2 g), urea (20 g), phenol red (0.012 g), per 1000 mL, pH 6.8.

Procedure

1. Streak the surface of a urea agar slant with a portion of a well-isolated colony or inoculate test organism on urea broth containing phenol red as the indicator.
2. Incubate the urea agar slant or urea broth at 37°C for 24-48 hours.
3. Examine the development of pink colour.
4. In case of unknown result incubate the tube for 7 days to check for slow urease production.

                               

1. Proteus spp
2. Corynebacterium spp
3. Helicobacter pylori
4. Brucella spp

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Methyl Red Test (MRVP broth) 

Procedure: Inoculate a loopful of bacteria into MRVP broth. Incubate 3 to 5 days.

 Description: Mixed acid fermentation -Many gram-negative intestinal bacteria can be     differentiated based on the products produced when they ferment the glucose in MR-VP medium. Escherichia, Salmonella, andProteusferment glucose to produce lactic, acetic, succinic, and formic acids and CO2, H2, and ethanol. The large amounts of acids produced lowers the pH of the medium -Methyl red (a pH indicator) will turn red when added to the medium if the organism was a mixed acid fermenter. Many of these organisms also produce gas.

Interpretation: After incubation: The broth must be turbid. A clear broth indicates that your organism did not grow and cannot be tested. Remove 1 ml of broth and place into a sterile tube before performing the methyl red test ifyou are going to use the same broth for the VP test. Add 3-4 drops of methyl red to the original broth. DO NOT SHAKE THE TUBE. A positive result has a distinct red layer at the top of the broth. A negative result has a yellow layer.


Voges-Proskauer Test

Procedure: Inoculate a loopful of bacteria into MRVP broth. Incubate 3 to 5 days.

Description: Organisms that are negative in the methyl red test may be producing 2, 3 butanediol and ethanol instead of acids. These non-acid products do not lower the pH as much as acids do.Enterobacter, Serratiaand some species of Bacillus produce these substances. There is no satisfactory test for determining production of 2, 3 butanediol. A precursor of 2,3 butanediol called acetoin can be detected with Barritt's reagent.

Interpretation: After incubation: Read the VP test when you have good turbidity. A clear broth indicates that your organism did not grow and cannot be tested. Barritt's reagent A (VP A) contains naphthol and Barritt's B (VP B) contains KOH. Test 1 ml of your culture from the MRVP broth. If you have already conducted the methyl red test, you should have already placed 1 ml of untested broth in a sterile tube. If you haven’t done this, do so now. Add the entire contents of the VP A reagent (15 drops) and 5 drops of the VP B reagent to the 1 ml of your broth culture. SHAKE WELL. This reaction will take a few minutes before you will see a color change. SHAKE the tube every few minutes for best results. With a positive reaction the medium will change to pink or red indicating that acetoin is present. With a negative reaction the broth will not change color or will be copper colored. Wait at least 15 minutes for color to develop before calling the test negative.





Citrate Test (Simmon's Citrate Slant)

Procedure: Streak a loopful of bacteria onto a citrate agar slant, do notstab the butt. Incubate 24 to 48 hours, longer forBacillus species. Incubate with a loose cap.

Description: Simmon's citrate agar tests for the ability of an organism to use citrate as its sole source of carbon. This media contains a pH indicator called bromthymol blue. The agar media changes from green to blue at an alkaline pH.

Interpretation: After incubation: A positive reaction is indicated by a slant with a Prussian blue color. A negative slant will have no growth of bacteria and will remain green.