Basic principle of specimen collection

#sheet (12). made by: marah marahleh corrected by: wael abu-anzeh date 27/10/2016

Basic principle of specimen collection "This is going to be the last lecture in the first exam." We talked previously about the sample collecting process, the right volume, the methodology, quality of specimen which are all factors that affect the identification process. Specimen Collection Guidelines Blood culture Disinfect skin with alcohol and iodine, blood culture media set (aerobic and anaerobic, bottles) or vacutainer tube with SPS( sodium polyanethol sulfonate) /adults, 20 ml per set; children 5 to 10 ml per set Body fluids (Abdominal, amniotic, ascites, bile, joint, pericardial, pleural), disinfect skin before needle aspiration sterile, screw-cap tube, 1 ml Cerebrospinal fluid Disinfect skin before aspiration, use sterile screw-cap tube/bacteria 1 ml, fungi, 2 ml, AFB 2 ml, virus 1 ml The volume should be 1 ml, less than one ml won't be accurate and the technician have the right to reject the sample ( if any of the criteria of sample collecting isn't met, the technician have the right to reject the sample otherwise it will give false result ). We also talked about blood culture, body fluid, CSF(cerebrospinal fluid ) and the volume needed for every analysis if we're looking for bacteria, fungi, AFB( acid fast bacilli) or viruses. Ear 1. Inner ear: clean ear canal with mild soap, aspirate fluid, with needle if eardrum intact; Or use swab if, eardrum ruptured, sterile, screw-cap tube or anaerobic transport system 2. Outer ear : remove debris or crust from ear canal with saline moistened swab;

rotate swab in outer canal, swab transport system Ear: 1) inner ear infection: more complicated, the physician collects the sample, the needle used is the typical syringe not the 10 ml or 5 ml that is used for blood samples if the eardrum is intact but if the eardrum is raptured we use ear swab we draw the sample form the pus in small tubule. We use anaerobic system if we are looking for anaerobic bacteria. For anaerobic bacteria we don t use the same method for aerobic bacteria if the physician suspects that there is anaerobic infection mainly in the inner ear bacteria, then the sample should be kept in an anaerobic transport system. 2) Outer ear: easily taken by the technician after removing the debris or crust from the external ear canal with normal saline. The swab should be moisturized before the sample is taken. What is the meaning of transport media? Swab is not directly taken to the lab it is put in a transport media which is a tube that contain agar to maintain the viability of the bacteria ( exact number ) during transportation. Sometime we need to transport the sample because the clinic where the sample is taken is far away from the lab. The agar used in the transport system is not nutritional and it is not used for the multiplication of the bacteria. The transport media contain suppressant media for normal flora and preservative to maintain the viability of the bacteria. Feces SAMPLES - Collect directly into container, avoid contamination with urine, clean, leak proof container or enteric transport system. - A rectal swab can be submitted for bacterial culture but it must show feces. A single specimen is not usually sufficient to exclude bacteria or parasites.

- if a bacterial infection is suspected, three specimens should be collected, one a day for 3 days. - if parasites are suspected, three specimens collected within 10 days should be sufficient for microscopic detection of ova and parasites. - The newer methods detect parasite antigens, and one sample is usually sufficient. -commercial systems are available with preservatives for bacteria and parasites. The appropriate ratio of stool to preservative is 1:3 Feces sample: for gastrointestinal tract infection we take either rectal swab or feces sample. rectum. Rectal swab: from the )مسحات شرجية( We should not use carton container for watery stool (diarrhea) because it contain fiber that will absorb the water and leakage will occur. so we use plastic container. Three states of the Stool: Formed Semiformed Watery (diarrhea) For formed or semiformed stool we can use the fiber container (carton) but for watery stool we have to use a plastic container. A single specimen or swab does not exclude the infection. Parasite: microscopic exam is important. Microscopic exam is not very important in bacteria sample but culturing is important Microscopic exam culturing biochemical tests Question by the Dr: if we are looking for bacteria the microscopic examination is not necessary on the other hand if we are looking for parasite infection it is important?

In the intestine we have normal flora (one of them E.coli) how can we recognize if this E.coli is toxigenic or cause food poisoning etc.. All of them are gram negative so we can't recognize them in the smear, it does not give any indication, but parasite are not normal flora and are recognizable. There are other methods to detect parasite in stool depending on: 1) Serology: (antigen / antibody) reaction. 2) Molecular polymerize reaction. Sometime we need to put the stool (or urine) in preservative media but other samples do not need preservation. The ratio of Bolic acid: for urine or stool sample preservation is 1:3. Fungal scrapings - Wipe nails or skin with alcohol, use clean, screw-cap container. A) hair/nails/skin hair: 10-12 hairs with shaft intact b) nails: clip affected area c) skin: scrape skin at outer edge of lesion Hair should be extracted with it's follicle (the area would have pus, very soft and the hair is easily removed). Genitalia A. Cervix/vagina: Remove mucus before collection; do not use lubricant on speculum; swab endocervical canal or vaginal mucosa, swab transport system or JEMBEC transport system Genitals: most samples are HVS (high vaginal swab). Vagina/cervix sample: taken by the gynecologist not by the technician by using speculum that is inserted inside the vaginal canal then the OS ( opening of the cervix) will appear then we clean the area with cotton swab to remove all excretion and debris then we insert another clean swab inside the OS. If there is a case of gonorrhea we should use JEMBEC transport media (plate special for isolation of pathogenic Neisseria which causes gonorrhea).

JEMBEC transport: rectangular in shape containing chocolate agar and special tablet (CO2 generating tablet) which is needed by the capnophilic bacteria ( 5%-10%) co2. B. Urethra : Flexible swab inserted 2-4 cm into urethra for 2-3 sec or collect discharge, swab transport system or JEMBEC transport system Lesion/wound/abscess Wipe area with sterile saline or alcohol, superficial swab along outer edge, swab transport system, deep aspirate with needle and syringe, anaerobic transport system urethra : male sample, if the male suffer from gonorrhea there is certain swab ( not the normal swab that is used) consisting of a fine wire containing a small swab which is inserted inside the opening of the urethra, sometime there is extra discharge produced extracellular from the male genital tract and sometimes there is none so we can insert the swab inside the urethra, get the sample and directly striking on jembec transport system "chocolate agar". For anaerobic infection: a deep aspirate with syringe because we have mixed infection and there is too much pus. jembec agar for Neisseria ( appear small shiny colonies) provide two factors that affect the growth of Neisseria : 1)X factor 2)V factor. "We will take about them later on"

Respiratory: 1) upper respiratory tract infection (sore throat, tonsillitis, pharyngitis) 2) Lower respiratory tract infection (pneumonia, bronchitis) Respiratory tract: Lower bronchial specimens - sputum, rinse mouth or gargle with water, instruct to cough deeply into container. Or the patient should rinse the mouth with water and expectorate with the aid of a deep cough directly into a sterile container (expectorated sputum). - Patients with dentures should remove the dentures first. A single specimen should be adequate for detection of bacterial LRT infection. If fungal or mycobacterial infections are possible, three separate early morning specimens (collected on successive days) are appropriate. - Specimens may be collected through aerosol-induction in which the patient breathes aerosolized droplets of a solution that stimulates cough reflex (induced sputum). If we are talking about lower respiratory tract specimen: then we are talking about sputum which is different form the saliva. (It's difficult specimen to take, if there is any contamination of normal flora from the upper respiratory tract then the sample is rejected) the sample should be obtained from deep. Pneumonia in the case of tuberculosis the media used is Lowenstein Jensen media where the patient can spit on. Pneumonia ( streptococcus pneumonia, haemophilus influenzae..) : a single specimen should be adequate ( from the lower respiratory tract ). Fungal infection such as histoplasmosis capsulatum, Cryptococcus neoformans and mycobacteria tuberculosis three sample on three successive days.. NAOH: is inhaled which cause irritation in the respiratory tract and cause cough reflex "induced-sputum" Respiratory tract: urt 1. Nasal : Insert premoistened swab with sterile saline 1 inch into nares, swab, use transport system 2. Nasopharynx : Insert flexible swab through nose into posterior nasopharynx, rotate for 5 sec, swab transport system or direct inoculation to media

3. Throat Swab, Posterior pharynx, tonsils, and inflamed areas, swab transport system Tissue: Disinfect skin; do not allow tissue to dry out; if necessary, moisten with sterile saline, use transport system or sterile screw-cap container. We take the sample from the area which appears inflamed where redness, pus and ulcers are observed. If there is tissue ( not swab ) inflammation scraps are taken and put in a container with normal saline to keep the tissue moist then transport it to the lab and do homogenization and divide it to small pieces then we use centrifusion and take the superlative and culture it on agar.

Lumber puncture : taken by the physician between L3-L4 or L4-L5, this is the most dangerous and valuable sample so we have to be sure there is no contamination at all,three samples should be taken: 1)for biochemistry tests 2) Hematology 3) Microbiology And that's because Not only the presence of microorganism is important but the levels of glucose and protein in the CSF is important too. Urine SAMPLES 1. Clean-catch midstream: Clean external genitalia; begin voiding and after several ml have passed; collect midstream without stopping flow of urine, sterile, screwcap container, collect 2-3 ml. The first portion of the urine flow washes contaminants from the urethra, and the midstream portion is more representative of that in the bladder. Urine: the most famous, widely used sample is the clean catch midstream sample (early morning midstream) because in the early morning the levels of microorganisms are at their highest and the patient drink solution and water during the day so the levels are lowered after the morning. The first 1/3 of the urine is excluded because it contains the normal flora in the urethra and first part of the bladder so we take the second 1/3 which contains the pathogens microorganism. The urine is collected in special screw-cup container with graduation. We do both micro and macroscopic exam (macro: the color, turbidity, glucose and protein level using strips) then culturing. The contamination level is 24%. 2. Catheter Clean urethral area, Insert catheter, and allow first 15 ml to pass; then collect remainder sterile, use screw-cap container or urine transport kit

Catheter clean urethral area: for people with bed rest or had gone under surgery and can t go to the bathroom. Catheter: from urethra opening to plastic bag for collection of the urine and at the end of the day the bag is removed. This method usually contains a lot of contamination. 3. Indwelling catheter, Disinfect catheter collection port, aspirate 5-10 ml with needle and syringe, use screw-cap container or urine transport kit Indwelling catheter: catheter is fixed in the bladder we draw the sample using syringe aspirate (5-10 ml) 4. Suprapubic aspirate, Disinfect skin, aspirate with needle and syringe through abdominal wall into full bladder, use sterile, screw-cap container or anaerobic transport system Superpubic aspirate: from the bladder ( upper genetalia ) taken by the physician by syringe. (Need to drink lots of water) Mostly used in babies who is under two years old with urinary tract infection. The level of contamination is the lowest between all methods (1%).because it is taken directly from the bladder and does not pass throw urethra.

Patient specimens or culture isolates must be TRIPLE PACKAGED BEFORE BEING SHIPPED. The material is placed into a Primary receptacle that must be watertight. Absorbent material is placed around the primary receptacle, and it is then placed into a Secondary container that is also watertight. The secondary package is sealed and placed into a sturdy Outer container constructed of fiberboard. Specific instructions must be followed for labeling the container as hazardous material. Specimen transport: when the specimen is taken in the clinic and need transportation to the lab. The sample should be kept in 3 containers and label the last container the hazardous material considering that any sample is infectious. When the sample reaches the lab how the technician deals with it: four levels

Specimen Priority A four-level scheme of prioritization may be used based on the critical nature of the specimen or potential for specimen degradation. Level 1: Critical/invasive ( amniotic fluid, blood, CSF, heart valves, pericardial fluid should deal with it immediately Level 2: Unpreserved ( body fluids, bone, drainage from wounds, feces, sputum, tissue) feces not preserved. Level 3 : Quantitation required (catheter tip, urine, tissue for quantitation) Level 4 : Preserved ( feces in preservative, urine in preservative, swabs in holding medium, (aerobic and anaerobic) need transport media. Otherwise the sample is put in the refrigerator not the freezer and for 24 hour maximum. Macroscopic Observation Notations from the macroscopic observation should include the following: stool consistency (formed or liquid) also the smell ( strong smell of stool is an indication of giardia infection caused by parasites ) and color. blood or mucus present volume of specimen : important for semen sample ( should be a specific volume otherwise there is abnormality ). fluid: clear or cloudy "if the urine is cloudy this indicate that there is infection " areas of blood and mucus are selected for culture and direct microscopic examination. Anaerobic cultures may be indicated if gas, foul smell, or sulfur granules are present. The diagnosis is evident if adult helminths or tapeworm proglottids are present in the specimen.. infection Progolottids : parasitic :ديدان شريطية

Microscopic Observation A direct microscopic examination is a useful tool that provides rapid informations (1) it can be used to determine the quality of the specimen. Sputum specimens that represent saliva rather than lower respiratory secretions can be determined by the quantitation of WBCs or epithelial cells. (2) it can give the indication of the infectious process involved. Gram stain of a sputum specimen revealing WBCs and Gram-positive diplococci is indicative of streptococcus pneumoniae. (3) the routine culture workup can be guided by the results of the smear. (4) it can dictate the need for nonroutine or additional testing. The presence of fungal elements in a specimen for bacterial culture will alert the technologist to notify the physician to request a fungus culture. Sometime we have fastidious bacteria it s not necessary to use fastidious plate we use plates for all purposes that provide the lowest nutritional needs but using gram stain we can know if we have fastidious sample from the shape of the bacteria such as haemophilus influenzae rod, filament.. so we use chocolate agar or agar that contain NAD(factor V) and hemin (factor X). Fungal media is completely different from bacteria media and is recognizable because the size is larger and the shape (filaments, hypha...) from the gram stains. Culture Media Selection - The selection of media to inoculate is based on the type of specimen submitted and the organisms likely to be involved in the infectious process. - Specimens in which fastidious pathogens are more likely involved require media with appropriate nutrients to aid in their recovery. - Specimens that are collected from a site containing normal biota will require

types of media to diminish the normal biota while allowing the pathogens to be detected. Such as in case of salmonella we use bismuth sulfite agar which inhibit the growth of normal flora and allow only salmonella to grow. The routine primary plating media include Nonselective agar plate ( for all organism) Enriched medium for fastidious organisms Selective and differential medium for enteric gram negative bacilli for most routine bacterial cultures ( MacConkey agar) Selective medium for gram-positive organisms for specimens in which mixed grampositive and gram negative bacteria are found.