1. Laboratory diagnoses of infections agents

2. Blood sample
Accuracy of any result depends upon the quality of the specimen.
Proper identification of the specimen are extremely important.
Each specimen should be clearly labeled with patients first and last name as it appears on the test request form. Additional desirable information on the specimen includes (1) date and time of collection, (2) account number and (3) specimen type (e.g. serum or plasma)

3. Specimen collection Blood samples
Detection of microorganisms by culture of blood is essential in the diagnosis of
bloodstream infections, including infective endocarditis
infections presenting as pyrexia of unknown origin,
prosthetic material infections and
Intravenous catheter infections.
Blood culture may also detect bacteraemia associated with primary infections such as pneumonia and septic arthritis. 
On the other hand, contaminated blood cultures can cause considerable diagnostic confusion and lead to unnecessary or sub-optimal antimicrobial therapy

4. Venipuncture Procedure
Several essential steps are required for every successful collection procedure.
1. Identify the patient
2. Assess the patients physical dispositions (i.e. diet, exercise, stress, basal rate)
3.Position the patient in a chair, or sitting or lying on a bed.
4. Wash hands.
5. Select a suitable site for venipuncture, by placing the tourniquet 3 to 4 inches above the selected puncture site on the patient.
6. Do not put the tourniquet on too tightly or leave it on the patient longer than 1 minute.
7. Next, put on non-latex gloves, and palpate for a vein.
8. When a vein is selected, cleanse the area in a circular motion, beginning at the site and working outward. Allow the area to air dry. After the area is cleansed, it should not be touched or palpated again.
9. Grasp the patient’s arm firmly using your thumb to draw the skin taut and anchor the vein. Swiftly insert the needle through the skin into the lumen of the vein. The needle should form a degree angle with the arm surface
10. When the last tube is filling, remove the tourniquet.
11. Remove the needle from the patient's arm using a swift backward motion.
12. Promptly send the specimens with the requisition to the laboratory.

6. Techniques to Prevent Hemolysis (which can interfere with many tests)
Mix all tubes with anticoagulant additives gently (vigorous shaking can cause hemolysis) 5-10 times.
Avoid drawing blood from a hematoma
If using a needle and syringe, avoid drawing the plunger back too forcefully. Air must not be pumped into the vein
Make sure the venipuncture site is dry before proceeding with draw.

7. Blood for smears Collection Handling and transport Capillary blood
make smear
fix with methanol or other fixative
Handling and transport
Transport slides within 24 hours
Do not refrigerate (can alter cell morphology)

8. Blood for cultures
For blood from young children, add 1-2 ml of blood into 20 ml of blood culture broth (approximately a 1:10 to 1:20 dilution).
For blood from adults, add 5-10 ml of blood into 50 ml of blood culture broth(approximately a 1:5 to 1:10 dilution).

9. Blood culture
Cultures for rapidly growing bacteria and yeast are usually incubated for 5 to 7 days.
Cultures for mycobacteria and slowly grow-ing fungi are held for as long as 42 days.
Many types of blood culture systems are available, including both manual and auto-mated. Each system utilizes a noninvasive method (i.e., colorimetric, fluorescent, or manometric methods for detecting CO2 or other gases) to monitor growth.

10. Blood Sample Centrifugation
It is recommended that serum be physically separated from contact with cells as soon as possible, with a maximum time limit of 2 hours from the time of collection.
In general, for a horizontal, swing-bucket centrifuge, the recommended spin time is 10 minutes. For a fixed-angle centrifuge, the recommended spin time is 15 minutes.
Tubes tops should remain closed at all times during the centrifugation process.

11. Swing-bucket centrifuge
Spin centrifuge

12. “balanced load”concept
Opposing tube holders must be identical and contain the same cushion or none at all.
Opposing tube holders must be empty or loaded with equally weighted samples (tubes same size and equal in fill).
If an odd number of samples is to be spun, fill a tube with water to match the weight of the unpaired sample and place it across from this sample.

13. Serum Collection Venous blood in sterile test tube
let clot for 30 minutes at ambient temperature
glass better than plastic
Handling
Place at 4-8oC for clot retraction for at least 1-2 hours

14. Serum Transport 4-8oC if transport lasts less than 10 days
Freeze at -20oC if storage for weeks or months before processing and shipment to reference laboratory
Avoid repeated freeze-thaw cycles
destroys IgM
To avoid hemolysis: do not freeze unseparated blood

15. Cerebrospinal fluid (CSF)
The collection of CSF is an invasive procedure and should only be performed by experienced personnel under aseptic conditions.
If bacterial meningitis is suspected, CSF is the best clinical specimen to use for isolation, identification, and characterization of the etiological agents.
Suspected agents should include N. meningitidis, S. pneumoniae, and H. influenzae and other pathogens in some cases.

16. Cerebrospinal fluid Collection
Ensure that the patient is kept motionless during the lumbar puncture procedure, either sitting up or lying on the side, with his or her back arched forward so that the head almost touches the knees in order to separate the lumbar vertebrae during the procedure
Disinfect the skin with 70% alcohol to clean the surface and remove debris and oils. Allow to dry completely.
Position the spinal needle between the 2 vertebral spines at the L4-L5 level. Accurate placement of the needle is rewarded by a flow of fluid, which normally is clear and colorless.

18. Remove CSF (1 ml minimum, 3-4 ml if possible) and collect into sterile screw-cap tubes.
If 3-4 ml CSF is available, use 3 separate tubes and place approximately 1ml into each tube.
Withdraw the needle and cover the insertion site with an adhesive bandage. Discard the needle in a puncture-resistant, autoclavable discard container.

19. Handling and transportation
Transport the CSF to a microbiology laboratory within 1 hour for culture and analysis.
If that is not possible, inoculate CSF into T-I medium (T-I is a biphasic medium that is useful for the primary culture of meningococci and other etiological agents of bacterial meningitis(S. pneumoniae and H. influenzae) from CSF. It can be used as a growth medium as well as a holding and transport medium)
If T-I is not available, incubate CSF at 35-37°C with ~5% CO2 and store in an approved location if the laboratory is closed.

21. Throat swab (posterior pharyngeal swab)
WHO/CDS/EPR/ARO/2006.1
Hold tongue away with tongue depressor
Locate areas of inflammation and exudate in posterior pharynx, tonsillar region of throat behind uvula
Avoid swabbing soft palate; do not touch tongue
Rub area back and forth with cotton or Dacron swab

22. Nasopharyngeal swab Tilt head backwards
Insert flexible fine-shafted polyester swab into nostril and back to nasopharynx
Leave in place a few seconds
Withdraw slowly; rotating motion
WHO/CDS/EPR/ARO/2006.1

23. Naso-pharyngeal aspirate
Tilt head slightly backward
Instill ml of sterile normal saline into one nostril (A nostril is one of the two channels of the nose)
Use aspiration trap
Insert silicon catheter in nostril and aspirate the secretion gently by suction in each nostril

24. Sputum
Collection
Instruct patient to take a deep breath and cough up sputum directly into a wide-mouth sterile container
avoid saliva or postnasal discharge
1 ml minimum volume

25. Respiratory samples Handling and Transport
All respiratory specimens except sputum are transported in appropriate media
bacteria: Amie’s or Stuart’s transport medium
viruses: viral transport medium (VTM)
Transport as quickly as possible to the laboratory to reduce overgrowth by oral flora
For transit periods up to 24 hours
ambient temperature for bacteria
4-8°C for viruses

26. Stool samples
The purpose of a stool culture (STC) is either to identify the causative agent of diarrhea, or to detect the bacterial carrier state in a patient
"stool culture for pathogens” includes screening for the presence of Salmonella, Shigella, Arizona,Edwardsiella, Campylobacter, shiga toxin producing E. coli, and for a predominance of Staphylococcus aureus, Pseudomonas, or yeast

27. Type of Stool samples Freshly passed stool samples Rectal swabs
avoid specimens from a bed pan
Rectal swabs
convenient
adapted to small children, debilitated patients and other situations where voided stool sample not feasible

28. Collection process
A single properly collected specimen is usually enough to identify the cause of acute bacterial diarrhea. To detect a carrier state, single specimens for three consecutive days are recommended. Only one specimen per patient per day will be accepted.
All stool specimens for culture should be submitted in a transport media

29. Stool specimens should be collected in a clean, dry container.
Stool specimens should not be contaminated with water, urine, barium, or mineral oil.
Add specimen to each vial until fluid level reaches the fill-to-here line. Do not overfill the vials.
Note specimen consistency on the outside of the vial
Liquid = of pourable consistency
Soft = having no shape
Formed = having a definite shape

30. Transportation of Stool Sample
The transport media is designed to maintain pH levels because some pathogens are sensitive to the pH changes associated with normal bacterial metabolism.
Currently, the transport vial for culture has an orange cap and contains a red-pink fluid. If the fluid is yellow, do not use the vial; or if the fluid turns yellow after the specimen has been added, it is not acceptable for culture and must be recollected

31. Stool samples for viruses
Timing
within 48 hours of onset
Sample amount
5-10 ml fresh stool from patients (and controls)
Methods
fresh stool unmixed with urine in clean, dry and sterile container
Storage
refrigerate at 4oC; do not freeze
store at -15oC - for Ag detection,polymerase chain reaction (PCR)
Transport
4oC (do not freeze); dry ice for (Ag detection and PCR)

32. Stool samples for bacteria
Timing
during active phase
Sample amount and size
fresh sample and two swabs from patients, controls and carriers (if indicated)
Method
Cary-Blair medium
For Ag detection/PCR – no transport medium
Storage
refrigerate at 4oC if testing within 48 hours, -70oC if longer; store at -15oC for Ag detection and PCR
Transport
4oC (do not freeze); dry ice for Ag, PCR detection

33. Stool samples for parasites
Timing
as soon as possible after onset
Sample amount and size
at least 3 x ml fresh stool from patients and controls
Method
mix with 10% formalin or polyvinyl chloride, 3 parts stool to 1 part preservative
unpreserved samples for Ag detection and PCR
Storage
refrigerate at 4oC; store at -15oC for Ag detection and PCR
Transport
4oC (do not freeze); dry ice for antigen detection and PCR

34. Urine Sample Collection
Urinary exosomes containing apical membrane and intracellular fluid are normally secreted through the urine
may carry protein markers of renal dysfunction and structural injury.

35. First Morning Specimen : This is the specimen of choice for urinalysis and microscopic analysis, since the urine is generally more concentrated (due to the length of time the urine is allowed to remain in the bladder) and, therefore, contains relatively higher levels of cellular elements and analytes such as protein, if present. Also called an 8-hour specimen.

36. Collection and processing
We collected usually first and then second morning spot urines from healthy volunteers. Protease inhibitors were added
Samples were stored at 4, -20, and -80°C for one week or 7 months.
Samples were thawed with and without extensive vortexing
3 fractions were isolated:
urinary sediment,
urinary supernatant, and
urinary exosome fraction

37. Special consideration
Midstream Clean Catch Specimen is the preferred type of specimen for culture and sensitivity testing because of the reduced incidence of cellular and microbial contamination. 
Urinary exosome-associated proteins were not detected in urinary sediment or supernatant fractions.
Protease inhibitors prevented degradation of exosome-associated proteins.
Freezing at -20°C caused a major loss in urinary exosomes compared to freshly collected urine.
In contrast, recovery after freezing at -80°C was almost complete (86%).
Extensive vortexing after thawing resulted in a markedly increased recovery of urinary exosomes in urine frozen at -20°C or -80°C, even if frozen for 7 months. 

38. Case investigation form
Patient information
age (or date of birth), sex, complete address
Clinical information
date of onset of symptoms, clinical and immunization history, risk factors or contact history where relevant, anti-microbial drugs taken prior to specimen collection
Laboratory information
acute or convalescent specimen
other specimens from the same patient

39. Biosafety: protect the patient
Use single use equipment Disinfect Work in a clean, dedicated area

40. Biosafety: protect yourself
Use personal protective equipment
disposable gloves
laboratory coats / gown
mask
protective eyewear / face shields if procedure is likely to generate aerosols
If no sharps container: collect sharps immediately to prevent needle-stick injury
Have first aid kit readily accessible
Do not reuse contaminated equipment