This test can be performed with the use of several media including Triple Sugar Iron (TSI), Kligler's Iron Agar (KIA), and SIM medium. 

An iron compound and a sulfur compound are included to test for the production of hydrogen sulfide gas.  Hydrogen sulfide is produced if the sulfur compound is reduced by the bacterial strain.  This happens when the strain either degrades the amino acid cysteine during protein degradation, or when anaerobic respiration shuttles the electrons to sulfur instead of to oxygen.  In either case H2S is produced (hydrogen sulfide gas) which reacts with the iron compound to form the black precipitate of ferric sulfide.  The black color acts as an indicator for the presence of hydrogen sulfide.  (If the tube becomes largely black, it may be difficult to read the tube for other tests.)


Procedure:

1. Refer to the medium that you are using for the procedure as they differ by medium.
2. Observe the medium in the tube and record its color and appearance in your notebook.

Results:

1. Look for the occurrence of H2S production (black coloration), comparing your tube to an uninoculated control if needed.
2. The black coloration may mask the color of the the tube, especially the lower end.
3. Make your interpretations and record your results and interpretations.

Instructor Notes:
TSI is less sensitive in detecting the production of hydrogen sulfide gas than other similar tests (SIM media, Kligler's Iron Agar, etc.).  So, it is possible to observe the production of hydrogen sulfide in another test and not observe it in TSI.  The sucrose in TSI may suppress the formation of hydrogen sulfide.  Both TSI and Kligler's Iron Agar also use ferrous sulfate to detect the production of H2S, and this detector is less sensitive than other hydrogen sulfide detectors.  TSI has an added sulfur source (sodium thiosulfate), but sulfur may also be obtained from certain amino acids present in the protein in the media.

Laminar Flow Hood (Optional).
Vacuum Pump with Rubber Tube.
Suction Flask 1 L.
Nitrocellulose Filter, Pore Size: 0.45 µm.
Microbiology Media.
Sterile Distilled Water.
70% Ethanol.
Flame.
Forceps.
Incubator.
Autoclave.
Tissue Paper.
Sterile cups or Sterile bags.
Suction Funnel.
Wet piece of tissue paper by 70% ethanol and wipe the valve of the tank.
Open the valve to let some water out to erase any excess of ethanol then close it.
Open the sterile bag or sterile cup in the same time open the valve and take 100 ml then close the valve and the bag / cup.
Transfer the sample to Microbiology lab immediately.
Water Samples can be stored in refrigerator at 4°C to inhibit the growth and the rise in microbial numbers.

 Aglütinasyon, uygun bir sıvı ortamda, partiküler formdaki antijenlerle antikorların  bağlandıktan sonra kompleksler oluşturarak bir arada kümelenmesidir. Bazı mikroorganizma veya hücre tiplerine karşı bağışıklık kazandırılmış serumlarda meydana gelen antikorlar bu mikroorganizma veya hücrelerle tekrar karşılaştığında onları, kümeler halinde birbirlerine yapıştırarak yani aglütine ederek etkisiz kılar. Genellikle enfeksiyonlarda antikorlar birinci haftadan sonra kanda görülmeye başlar. 

Birçok hastalığın teşhisinde, kan gruplarının belirlenmesinde bu aglütinasyon özelliğinden yararlanılır. Aglütinasyon testleri mikroskopta, tüpte ve lamda yapılır. Aglütinan antikorlar, genel olarak IgM ve IgG karakterindedir. 
Aglütinasyon Test Sonuçlarını Değerlendirirken Dikkat Edilecek Hususlar 
➤Normal serumun içinde düşük titrede antikor bulunacağından dilüsyonların yüksek titrelere kadar yapılmasına dikkat edilmelidir. 
➤Özellikle hastalığın akut safhasında ve 7-10 gün sonra alınan çift serumlarla testler çalışılıp titrenin yükselip yükselmediği kontrol edilmelidir. 
➤Daha önce yapılan aşılanmanın kanda belli oranda antikor ihtiva edeceği göz önünde bulundurarak teşhisin buna dikkat edilerek yapılması gerekir. 
➤Antijen benzerliğiyle bazı bakteriler arasında çapraz reaksiyonlar olur. Bu gibi durumlarda yüksek titrede reaksiyon veren antijen hastalık etkeni olarak kabul edilir. 
➤Bazen yeni bir enfeksiyon hastalığı önceden geçirilmiş ve antikorları kandan kaybolmuş önceki hastalığa ait aglutininlerin ortaya çıkmasına neden olur. 
➤Serumun uygun şekilde sulandırılmasına dikkat edilmelidir. 

• Antigen (Rose bengal dye special technique stained with buffered saline to Brucella abortus bacteria are the standard suspension.) 
• 1.5 cm diameter shallow pits with plates  
• Patient serum 
• Toothpick 

Technical  
➤ One of the plate wells, 0.03 ml of patient serum is placed. 
➤ 0.03 ml of patient's serum on antigen mixed thoroughly put 
➤ a toothpick spread over an area of 1.5 cm diameter with good stirring. 
➤ Obtained or converted by circular movement on a rotator for 4 minutes. 
➤ While the end results are read.

Brucella abortus'un tanısını koymaktır.
Araç Gereçler

• Antijen (Rose bengal boyası ile özel teknikle boyanan, Brucella abortus bakterilerinin tamponlu tuzlu sudaki standart süspansiyonlarıdır.) 
• 1,5 cm çapında yayvan çukurlar bulunan plaklar  
• Hasta serumu 
• Kürdan 

Teknik 
➤ Plak çukurlarından birine, 0.03 ml hasta serumu konur. 
➤ Hasta serumunun üstüne 0,03 ml iyice karıştırılmış antijen konur
➤ Bir kürdanla iyice karıştırılarak 1,5 cm çaplı bir alana yayılır. 
➤ Elde ya da bir rotatorda dairevi hareketler yaparak 4 dakika çevrilir. 
➤ Süre sonunda sonuçlar okunur.



Testin Değerlendirilmesi 
➤ Sonuçta iri tanecikli çökeltiler (aglütinasyon) oluşmuşsa; pozitif, 
➤ İnce tanecikli çökelti oluşmuşsa; kuşkulu,  
➤ Homojen görüntü oluşmuşsa; negatif olarak değerlendirilir. 

A negative stain may be used  similar to a basic simple stain or as a simple capsule stain. We will only use it as a simple capsule stain.  A capsule is an extracellular structure that some species make that is destroyed by normal staining procedures (especially heat fixing and washing).  Capsule staining is a gentle staining procedure that does not destroy the bacterial capsule.  This procedure stains the background but does not stain the bacterial capsule or the bacterial cell.  (Note, some other capsule stains use a second stain as a counter-stain which does stain the cell or even the capsule with another color.)

Purpose: to make the cells show up better (by adding higher contrast to the background) and to allow for viewing the capsule.




Procedure for a Simple Capsule Stains (or a Wet Mount using a Negative Stain):
➤ Do not start with a bacterial smear!  Set up your bacteria, two to four slides, two cover slips, a Bunsen burner, and your loop.  It is also a good idea to make a third slide containing a positive control (a bacterial strain that has a capsule) if one is available so that you may view a positive result.  (One may find bacteria with capsules by gently inserting a clean toothpick under your gumline next to a tooth.)
➤ Place a small drop of a negative stain (India Ink, Congo Red, Nigrosin, or Eosin) on your slides.  Congo Red is easier to see, but it does not work well with some strains.  India Ink generally works but it has tiny particles that display Brownian motion that must be differentiated from your bacteria.  Nigrosin may need to be kept very thin or diluted. Sterilize your loop and add an almost invisible amount of bacteria to one slide, smearing it in the dye.  You may spread your drop with the loop, or with a second slide that is brought into the stain drop and is then spread across the first slide.  It is recommended that you make more than one slide of a specimen, slightly increasing the amount of bacteria added to the second.
➤ Reflame your loop. 
➤ Add cover slips at a 45 degree angle to each slide.  You are essentially making a wet mount using a negative stain instead of water.
➤ First observe the slide with the greater number of bacteria, it is probably too many to work with, but look at it first to more easily observe your bacteria, look for an area of clearing caused by a  capsule around the cells.  Otherwise the dye should come right up to the cell.  When you think you know what you are seeing, switch to the slide with the fewer bacteria that hopefully will contain only a single layer of cells to be certain of your conclusions.
➤ If your bacteria are very motile, you may need to wait for them to die under the light before making your observations about a capsule.  You can turn up the light to speed this.
➤ Remember that a slide stained with a capsule stain contains live bacteria that should be handled with care and disposed of properly.



Procedure for a Dried Negative Stain:
We will not perform this procedure, it is detailed here just for your knowledge benefit.  Essentially, it is the similar to a Simple Basic Stain, except it uses a Negative Stain, and there are no washing and blotting steps.  Recall that negative stains are simple stains that do not stain the cell, they stain the background.

• Obtain a clean slide that has no fingerprints or dust on it (your instructor will inform you how to clean it if necessary.)
• Obtain a simple Negative Stain (India Ink, Congo Red, Nigrosin, or Eosin).  
• Place a small drop of a Negative Stain on one end of your slide.  
• Add a small amount of your bacterial culture to the drop and mix evenly without spreading.
• Now, bring a second slide held at a 45 degree angle into the stain drop and with this slide, spread the drop across the first slide.
• Dispose of the second spreader slide in disinfectant.
• Allow the slide to air dry.  Do not heat fix or wash.  View the slide under the microscope, a coverslip may be added if desired when using oil.
• Note, this procedure should not be used to look for capsules as the stain may shrink away from the cell during drying.

Widal Test

The main principle of widal test is that if homologous antibody is present in patients serum, it will react with antigen in the reagent and gives visible  agglutination in the tube. The antigens used in the test are H and O antigens of Salmonella Typhi.                                                   
The titre of the patient serum using Widal test antigen suspensions is the highest dilution of the serum sample that gives a positive result (Titers are expressed by the denominator only, for example 1:256 is written 256). The sample which shows the titre of 100 or more for O agglutinations and 200 or more for H agglutination should be considered as clinically significant (active infection).

Demonstration of 4-fold rise between the two is diagnostic. H agglutination is more reliable than O agglutinin. Agglutinin starts appearing in serum by the end of 1st week with sharp rise in 2nd and 3rd week and the titre remains steady till 4th week after which it declines.

Sitokrom oksidaz, bazı bakteriler tarafından sentezlenen bir enzimdir. Oksidaz testi bu enzimin aktivitesini ölçmeye yarayan bir testtir. Sitokrom oksidaz, fenilendiamin bileşiklerini okside ederek koyu mavi renkli indofenole dönüştürür. Neisseria ve Pseudomonas türlerinde oksidaz pozitif, Enterobacteriaceae türlerinde negatiftir.



Testin Yapılış Tekniği;
➤ Steril bir petri kutusu içine yerleştirilmiş kurutma kağıdına 2-3 damla oksidaz
ayıracı (dimetil veya tetrametil fenilendiamin dihidroklorid) damlatılır.
➤ Üzerine, öze ile şüpheli koloniden bir miktar alınarak sürülür.
➤ 10-60 saniye içinde koyu mavi renk oluşması, testin pozitif olduğunu gösterir.