**3. Important terms n takng examnaton sample**

As delay in the diagnosis of acute infection is unhealthful in terms of delay in the treatment and it is also dangerous that other persons may be infected due to more contact. To obtain rapid and correct etiological diagnosis is possible to take the examination material appropriately, to send it to the laboratory rapidly and to examine them in the laboratory well. Inability to produce the active germ is due to faulty examination material taking in general. Examination material should be taken by persons who know the purpose of such procedure and by being careful in the following subjects.

It should be especially noted that examination material should be taken before administration of any antibiotics or other chemotherapeutic medications. Pathogen bacteria may not exist in the purulence even 24 hours after antibiotic administration. If bacteria that chemotherapeutic substance inhibit can not be produced from the examination material during the administration, it may be produced several days after discontinuation of the medication.

Isolation and Identification of Salmonellas from Different Samples 129

containers to put stool. These are tubes made of glass which were closed by cork. A small spoon made of metal was put on the edge of cork seal. Purulence or mucous parts of stool is taken by this spoon and put into the tube and cork of the tube is closed. To send to far distances, this special tube is put into a cylinder shaped box and this box is put into a wooden box. Also, scoop shaped small spoons made of metal are used to take stool. The stool is taken by deep side of this spoon which is sterilized by wrapping to a paper and the spoon is put into a screw cap bottle. Especially, bottles with wider orifices are suitable to put

If culture of the stool will delay for several hours after taking, 1 g stool is mixed with 10 cm3 30% neutral glycerol solution in 6% saline buffer. It is seen that reaction is alkaline by pinkviolet color after addition of phenol red. If this mixture is kept for a period, reaction becomes acid and indicator shows yellow color; so this mixture is not used anymore. Glycerol solution prevents saprophyte bacteria to reproduce and mask pathogen bacteria.

> The solution is prepared as follows: 6% saltwater 70 cm3 Glycerol 30 cm³ Na2 HPO4 1 g 0,02% phenol red is added into this.

Prepared solution is divided into bottles as 10 cm3. The stool is taken into this bottle. Stool may be sent to a far laboratory by a filter paper to search *Salmonella* bacteria. For this, stool is applied on a filter or blotter as a thin layer and left for drying in the room. Two edges of the paper is folded by superimposing by a holder, stool is kept between the folded part. The paper is placed into a plastic envelope. Various samples which were prepared by this manner may be sent with a package prepared accordingly for mail. Such paper is cut into three parts in the laboratory. Suspension is made with salt water and it is smeared on appropriate medium in petri plate. Other parts are put into selenite and tetra

Stool sample which was taken into sterile containers or stool sample which was taken by swab rectally, the sample taken from bedpan or diaper of unconscious patient, and stool sample taken from baby diapers should be examined within at least one hour. If the sample is not examined within one hour, it may be stored in the refrigerator for three to four hours. Because, coliform bacteria which are dominant on the stool reproduce more in the room temperature and acidize the environment. This prevents reproduction of

Bean sized sample is taken into any of selenite or tetra thionate bouillon mediums and processed to pre-enrichment at 37°C for 3 to 4 hours in the incubator, then it is planted on SS, EMB, endoagar plates according to dilution planting technique and identification

Kidneys, ureter, bladder and most of the urethra are sterile areas in healthy human. Urethra includes a bacteria flora as far as 1,5-2 cm inside from the orifice both in women and men. In normal, the urine coming from upper sterile areas always contaminates more or less while

thionate bouillon, they are smeared on the petri plate after reproduction.

*Salmonella* bacteria as well as causes death.

**3.3 Microbiological examination of the urine** 

process is started by considering lactose negative colonies.

stool. Stool may be taken into glass like cardboards directly.

Examination material should be taken from the place where suspicious germ may exist. While taking sterile materials in normal such as urine, cerebrospinal fluid, special attention should be paid for contamination with outer germs. Sometimes, falling of several bacteria from outside causes not to obtain a result from the culture. The orifice of the tube or bottle should be singed when it is opened or before closing after material is put. The cap or seal of the tube or the bottle should be closed after removal without contacting any where. Although an extreme care is not necessary for materials including normal flora such as phlegm and stool, it is appropriate to take materials carefully by avoiding contamination from outside.

To obtain a successful result from the culture of the examination material, it should be taken within an appropriate period of the disease. In intestinal infections, bacteria may exist more in diarrhea period. It is more possible to isolate them within this period.

Furthermore, to take the examination material within certain times of the day may be important. To obtain a positive result by a culture made with the blood which was taken in febrile period is more likely. To obtain the phlegm in the morning is easier. The patient may expectorate more with a less effort.

No disinfecting or antiseptic are added into the examination material. If upper surface of the lesion is desired to be cleaned, a swam immersed into sterile saline or sterile cotton which is hold by a sterile holder is used. The material is taken after cleaned area dried.

Examination material should be sufficient to perform the examinations completely. It is not necessary to take excessive material. For example, when materials such as phlegm or stool are put more, they smear outside of the container. Particularly, a janitor who does not know the importance of the procedure may be infected by touching these containers. This should be prevented by continuous warnings and trainings.

The examination material should be sent to the laboratory as soon as possible and examined. Sensitive bacteria in the material of which examination is delayed die due to low temperature, effect of enzymes, drying or not being nourished. Saprophyte bacteria which may be present in the examination material reproduce at room temperature; to produce pathogenic bacteria is not possible as their count increases. Therefore, the material is stored in the refrigerator until the examination time when germs which are effected from cold are not searched. By this procedure, saprophytes do not reproduce and pathogens continue their vitality. Sometimes, mediums and required tools are carried near to the patient's bed and examination material is added immediately when taken.

Examination material should be sent to the laboratory early in the day. By this means, examination is possible within working hours of the laboratory. Laboratory should be notified one hour earlier for immediate examination.

#### **3.1 Taking examination material and putting in a suitable container**

It is extremely important not to contaminate with external germs for the material of which microbiological examination will be performed. The material which is taken in aseptic conditions should be put in a sterile container immediately and the cap should be closed. When germs in the examination material kept in hot conditions, they may reproduce and die in the cold. Therefore, the material which is taken to a sterile container should be implanted to the medium as soon as possible.

#### **3.2 Stool**

The stool that culture will be performed should be taken into a sterile container. By this means, contamination with bacteria in containers that stool was put before. There are special

Examination material should be taken from the place where suspicious germ may exist. While taking sterile materials in normal such as urine, cerebrospinal fluid, special attention should be paid for contamination with outer germs. Sometimes, falling of several bacteria from outside causes not to obtain a result from the culture. The orifice of the tube or bottle should be singed when it is opened or before closing after material is put. The cap or seal of the tube or the bottle should be closed after removal without contacting any where. Although an extreme care is not necessary for materials including normal flora such as phlegm and stool, it is

To obtain a successful result from the culture of the examination material, it should be taken within an appropriate period of the disease. In intestinal infections, bacteria may exist more

Furthermore, to take the examination material within certain times of the day may be important. To obtain a positive result by a culture made with the blood which was taken in febrile period is more likely. To obtain the phlegm in the morning is easier. The patient may

No disinfecting or antiseptic are added into the examination material. If upper surface of the lesion is desired to be cleaned, a swam immersed into sterile saline or sterile cotton which is

Examination material should be sufficient to perform the examinations completely. It is not necessary to take excessive material. For example, when materials such as phlegm or stool are put more, they smear outside of the container. Particularly, a janitor who does not know the importance of the procedure may be infected by touching these containers. This should

The examination material should be sent to the laboratory as soon as possible and examined. Sensitive bacteria in the material of which examination is delayed die due to low temperature, effect of enzymes, drying or not being nourished. Saprophyte bacteria which may be present in the examination material reproduce at room temperature; to produce pathogenic bacteria is not possible as their count increases. Therefore, the material is stored in the refrigerator until the examination time when germs which are effected from cold are not searched. By this procedure, saprophytes do not reproduce and pathogens continue their vitality. Sometimes, mediums and required tools are carried near to the patient's bed

Examination material should be sent to the laboratory early in the day. By this means, examination is possible within working hours of the laboratory. Laboratory should be

It is extremely important not to contaminate with external germs for the material of which microbiological examination will be performed. The material which is taken in aseptic conditions should be put in a sterile container immediately and the cap should be closed. When germs in the examination material kept in hot conditions, they may reproduce and die in the cold. Therefore, the material which is taken to a sterile container should be

The stool that culture will be performed should be taken into a sterile container. By this means, contamination with bacteria in containers that stool was put before. There are special

appropriate to take materials carefully by avoiding contamination from outside.

hold by a sterile holder is used. The material is taken after cleaned area dried.

in diarrhea period. It is more possible to isolate them within this period.

expectorate more with a less effort.

be prevented by continuous warnings and trainings.

and examination material is added immediately when taken.

**3.1 Taking examination material and putting in a suitable container** 

notified one hour earlier for immediate examination.

implanted to the medium as soon as possible.

**3.2 Stool** 

containers to put stool. These are tubes made of glass which were closed by cork. A small spoon made of metal was put on the edge of cork seal. Purulence or mucous parts of stool is taken by this spoon and put into the tube and cork of the tube is closed. To send to far distances, this special tube is put into a cylinder shaped box and this box is put into a wooden box. Also, scoop shaped small spoons made of metal are used to take stool. The stool is taken by deep side of this spoon which is sterilized by wrapping to a paper and the spoon is put into a screw cap bottle. Especially, bottles with wider orifices are suitable to put stool. Stool may be taken into glass like cardboards directly.

If culture of the stool will delay for several hours after taking, 1 g stool is mixed with 10 cm3 30% neutral glycerol solution in 6% saline buffer. It is seen that reaction is alkaline by pinkviolet color after addition of phenol red. If this mixture is kept for a period, reaction becomes acid and indicator shows yellow color; so this mixture is not used anymore. Glycerol solution prevents saprophyte bacteria to reproduce and mask pathogen bacteria.


0,02% phenol red is added into this.

Prepared solution is divided into bottles as 10 cm3. The stool is taken into this bottle.

Stool may be sent to a far laboratory by a filter paper to search *Salmonella* bacteria. For this, stool is applied on a filter or blotter as a thin layer and left for drying in the room. Two edges of the paper is folded by superimposing by a holder, stool is kept between the folded part. The paper is placed into a plastic envelope. Various samples which were prepared by this manner may be sent with a package prepared accordingly for mail. Such paper is cut into three parts in the laboratory. Suspension is made with salt water and it is smeared on appropriate medium in petri plate. Other parts are put into selenite and tetra thionate bouillon, they are smeared on the petri plate after reproduction.

Stool sample which was taken into sterile containers or stool sample which was taken by swab rectally, the sample taken from bedpan or diaper of unconscious patient, and stool sample taken from baby diapers should be examined within at least one hour. If the sample is not examined within one hour, it may be stored in the refrigerator for three to four hours. Because, coliform bacteria which are dominant on the stool reproduce more in the room temperature and acidize the environment. This prevents reproduction of *Salmonella* bacteria as well as causes death.

Bean sized sample is taken into any of selenite or tetra thionate bouillon mediums and processed to pre-enrichment at 37°C for 3 to 4 hours in the incubator, then it is planted on SS, EMB, endoagar plates according to dilution planting technique and identification process is started by considering lactose negative colonies.

#### **3.3 Microbiological examination of the urine**

Kidneys, ureter, bladder and most of the urethra are sterile areas in healthy human. Urethra includes a bacteria flora as far as 1,5-2 cm inside from the orifice both in women and men. In normal, the urine coming from upper sterile areas always contaminates more or less while

Isolation and Identification of Salmonellas from Different Samples 131

Proteus, Pseudomonas, Mimeae, Flavobacterium, Bacteroides, Listeria monocytogenes,

While CSF is taken, the area where puncture will be performed should be cleaned with iodine and alcohol to avoid contamination with flora bacteria on the skin. Sufficient material should be taken to be able to perform cellular, chemical and microbiological examinations. To take material, to put sterile screw cap tubes in lumbar puncture sets is more appropriate instead of cotton or plastic seal tubes. In cases that sufficient CSF can not be obtained, only required examinations should be requested first from the laboratory. CSF should be transmitted to the laboratory immediately and examined instantly. As CSF is regarded as materials that planting should be performed near the patient, if examinations can not be done within a very short period it should be stored in the incubator. The sample taken to isolate *Salmonellas* hould be taken into selenite f medium and it is kept under pre-incubation at 37°C for 3 to 4 hours, then planted on mediums such as SS, EMB, MAC KONKEY by dilution planting technique and lactose negative

Examination material is taken from ileum in suspicion of typhoid fever. Intestinal wall is cauterized by superheated spatula. A sterile swab is inserted from the hole opened and moved on the mucosa by rotating. The swab is removed and immersed into tetrahionate bouillon or selenite bouillon. The swab is rotated by pressing on the wall of the tube and a clouded fluid is obtained. Planting is performed from this fluid to Endo, SS, bismuth sulphite agar mediums which are used to produce *Salmonella* bacteria. Material is taken

The infectious agent from lesions such as abscess, wound and fistula are searched by

The purulence is taken from open lesions by a swab or a loop which is superheated

 If there is a closed abscess, upper surface is cleaned with tincture of iodine and alcohol and drying is waited. Abscess is opened, the first purulence is removed, the purulence appeared is taken by a swab, it is put into the tube without contacting the skin. Or, purulence is taken from the abscess by a sterile syringe or a Pasteur pipette and put into a sterile tube. Small pustules are pierced with a sterile needle after the upper surface is cleaned and the fluid appeared is taken by a Pasteur pipette, swab or

Liquids accumulated in cavities of pleura, pericardium and peritoneum and synovial and hydrocele liquids are sent to the laboratory to search for the bacteriological agent in case of

Serous liquids should be taken under sterile conditions and out into a sterile container. To prevent the coagulation by putting some of the liquid into a citrate solution or a beaded

Pasteurella multocida, Leptospira, Cryptococcus, Neoformans viruses.

colonies are identified.

**3.5 Autopsy material** 

**3.6 Purulence** 

from the bladder by the same way.

bacteriological examination. Taking Examination material:

a loop and examined.

and cooled.

**3.7 Serous liquids** 

bottle is appropriate.

infection.

passing from this region of the urethra. But this contamination is not over a certain bacteria count in a healthy person. By considering this feature of the urinary system and the urine, some criteria should be followed before taking the urine sample, while taking the sample and sending it to the laboratory for microbiological examinations.

Generally, urinary antiseptics and antibiotic drugs and excessive water should not be given to persons before taking the sample.

The form so called middle flow is the most common method applied in all hospitalized and polyclinic patients who are cooperated for taking urine sample. The most important subject that patients should know and should be informed in this method is genital region cleaning which is required to be performed before taking the urine sample. Because, bacteria reproduce over 105 criteria in urine samples taken from persons who do not care about genital area hygiene and cleaning, frequently in women and urinary infection is diagnosed inaccurately. No infection is found in urine samples of these persons after a well cleaning is performed. Soap and water are sufficient for cleaning. Persons should wash their hands first, and clean glans penis and orifice in men and genital areas, labias and the orifice of women from front to back and from up to down. The person will start to urinate just after this cleaning and take the urine which is in the middle of the urination stage, not the first or the last urine into a sterile tube or sterile bottle with a wide opening (as 5 to 10 ml) and close the cap immediately. The urine taken should be transmitted to the laboratory as soon as possible. If this is not possible, the urine should be stored in the refrigerator for a certain period.

The urine sample brought to the laboratory should be stored in the refrigerator or examined within 1 hour.

The other process that will be done with the urine sample is culturing.

The method used in almost every microbiology laboratory is to plant with a washing bottle or loop. Loops used for planting are fabrication products which are calibrated and they are expensive. Apart from this, they should be controlled whether their calibration was deteriorated by special methods. The most suitable for us is 1 ml serological pipettes with 0,1 calibrated.

The urine brought is planted as 0,1 ml on every petri plate (blood agar- EMB, endo, Mac Conkey etc.) by a pipette after mixing well and smeared to the plate by a sterile glass rod.

Plates put into the incubator (35-37°C) are removed after an incubation of 18 to 24 hours, and colony count is performed. The colony amount which is counted in every plate is multiplied with 10 and it is expressed as colony amount in ml. According to the values obtained, colony count between –10,000 is accepted as NORMAL, between 10,000-100,000 as SUSPICIOUS and over 100,000 as URINARY INFECTION.

Except these criteria, lactose negative colonies are evaluated and identified in EMB or ENDO AGAR; and *Salmonella* is diagnosed. In this case, this is accepted as an infectious agent without considering 100,000 ml/cfu bacteria count as we accepted as infectious agent.

### **3.4 Cerebrospinal fluid**

Bacteriological examination of cerebrospinal fluid is performed in cases that meningitis is suspected. Examination material taken under sterile conditions is sent to the laboratory as soon as possible. If the material will not be examined instantly, it is stored in the incubator to examine within one hour. Germs obtained from cerebrospinal fluid most:

Neisseria meningitidis, Diplococcus pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pyogenes, *Salmonella* escherichia, Proteus, Pseudomonas, Mimeae, Flavobacterium, Bacteroides, Listeria monocytogenes, Pasteurella multocida, Leptospira, Cryptococcus, Neoformans viruses.

While CSF is taken, the area where puncture will be performed should be cleaned with iodine and alcohol to avoid contamination with flora bacteria on the skin. Sufficient material should be taken to be able to perform cellular, chemical and microbiological examinations. To take material, to put sterile screw cap tubes in lumbar puncture sets is more appropriate instead of cotton or plastic seal tubes. In cases that sufficient CSF can not be obtained, only required examinations should be requested first from the laboratory. CSF should be transmitted to the laboratory immediately and examined instantly. As CSF is regarded as materials that planting should be performed near the patient, if examinations can not be done within a very short period it should be stored in the incubator. The sample taken to isolate *Salmonellas* hould be taken into selenite f medium and it is kept under pre-incubation at 37°C for 3 to 4 hours, then planted on mediums such as SS, EMB, MAC KONKEY by dilution planting technique and lactose negative colonies are identified.
