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Microbiology 2
Microbiology

LifeBridge Health - Department of Pathology

Microbiology
B. Specimen transport: packaging and mailing regulations for off-site delivery  
     
  General considerations  
       
  d) Place cultures of etiological agents in securely closed, watertight containers. Seal these primary containers tightly to prevent leakage during transport. Do not use petri dishes. Culture isolates must be subcultured onto a slant of appropriate media and tightly capped and sealed with parafilm.  
     
  e) Do not contaminate primary or secondary containers.  
       
  f) The secondary (outer) container must be seal-able and break resistant. USE TWO CYLINDER SYSTEM PROVIDED BY STATE HEALTH DEPARTMENT.  
     
  g)

Include the names, addresses, and telephone numbers of the sender and recipient laboratories on both the containers and the accompanying documents.  

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III. Collection Instructions for Different Anatomic Sites
   
  Any collection method requiring an invasive technique should be performed by a physician, and some specimen collection techniques should be performed only by a physician specialist with advanced training and skills. The specimen collection guidelines in this section are brief summaries of the procedures used for specimen collection and are not intended to be used as a step by step guide for obtaining specimens.
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A. Blood cultures
   
1. General considerations
   
  a) Number and timing
    Most cases of bacteremia are detected by using three sets of separately collected blood cultures. More than three sets of blood cultures yield little information. Conversely, a single blood culture may miss intermittently occurring bacteremia and make it difficult to interpret the clinical significance of certain isolated organisms.
   
   
  1. Acute sepsis
    Collect teo or three cultures from separately prepared sites prior to starting therapy.
  2. Endocarditis
    Acute: Obtain three blood cultures with three separate venipunctures over 1 to 2 hours, and begin therapy. Subacute. Obtain three blood cultures on day 1 (15 minutes or more apart). If all are negative 24 hrs later, obtain 3 more.
    Antimicrobial therapy 1 to 2 weeks before admission
    Obtain two separate blood cultures on each of three successive days.
  3. Fever of unknown origin
    Obtain two separate blood cultures at least 1 hour apart. If these are negative, then 24-36 hours later, obtain two more blood cultures 1 hour apart. The yield of information beyond four cultures is usually minimal.
   
  b) Volume of blood
    The volume of blood is critical because the concentration of organism in most cases of bacteremia is low, especially if the patient is on antimicrobial therapy. In infants and children, the concentration of organism during bacteremia is higher in adults, so less blood is required for culture.
   
   
  1. Children: 1 to 3 ml of blood per venipuncture
  2. Adults: 8 to 10 ml of blood per venipuncture
   
  c) Culture medium and culture technique
   
 
  1. General considerations
    (a) The culture medium should contain 0.025 to 0.05% sodium polyanetholesulfonate (SPS).
    Exception: In suspected cases of meningococcemia or gonococcemia, use medium without SPS. SPS inhibits the growth of some variants of Neisseria species. SPS is an anticoagulant that inhibits serum bactericidal activity against many pathogenic bacteria, inhibits phagocytosis, inactivates complement, and neutralizes lysozymes and the aminoglycoside class of antimicrobial agents.
    (b) The recommended volume ratios of blood to medium are from 1:3 to 1: 10.
  2. Media and techniques that are used for the isolation of aerobic and anaerobic organisms.
    (a) Bactec broth bottles: Standard 10 Aerobic 1F, Standard Anaerobic 1F, Plus +Aerobic 1F, Plus +Anaerobic 1F
  3. Media and techniques used for the isolation of fungi
    (a) Lysis centrifugation device (ISOLATOR; Wampole)
  4. Media and techniques used for the isolation of mycobacteria
    (a) Radiometric blood culture bottle (BACTEC Myco/ F Lytic).
 
2. Blood Collection
  Blood can be collected by venipuncture of peripheral veins or arteries, from intravascular catheters, or by heel stick. Observe universal precautions. Wear gloves.
   
  A) A minimum of two sets of blood cultures< should be obtained on two different occasions within a 24-hour period. The interval between blood cultures should not be less than 10 minutes apart (depending on the clinical presentation).
   
  B) For patients suspected of having bacterial endocarditis, a minimum of three (3) and a maximum of five (5) blood cultures should be obtained within a 48-hour period, at intervals not less than 10 minutes apart.
   
    Specimen Collection:
   
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The 7 steps to Blood Culture Collection…

 
  1. Examine the blood culture bottles

2. Mark bottles for appropriate specimen collection amount
3. Disinfect the bottle tops
4. Appropriate Identification the patient
5. Apply the tourniquet
6. Skin Preparation
7. BD Butterfly Method
   
   
 

Step 1: Examine the blood culture bottles
 
  Discard the bottles if any of the following is observed:
 
  -  Contamination, damage, or deterioration
  -  Cloudy or turbid MEDIA
  -  More or significantly less than 2 inches of liquid media is inside the bottle
  -  Media is expired
  -  Sensor is the wrong color. The sensor is located on the bottom of the bottles and is used by the blood culture instrument to detect growth.
   
 
   
   
 

Step 2: Mark bottles for appropriate specimen collection amount
   
  The bottles are marked in 5 mL increments along the label. The only way to ensure that the appropriate amount of sample is collected is to mark off the broth level and indicate the fill level.

8-10 mL of blood per bottle is recommended for adults.
   
 
   
   
 

Step 3: Disinfect the bottle tops
   
  Remove the protective caps from the Bactec blood culture bottles and disinfect the top of each bottle septum with 70% alcohol and allow to air dry. Use 1 alcohol wipe for each bottle septum.
Do Not Use Providine Iodine on the Bactec blood culture bottles. After disinfecting the bottle septums, do not retouch the bottle tops.
   
 
   
   
 

Step 4: Identify the Patient
   
  Appropriate identification of patient and explanation procedure to patient are required before proceeding.
   
   
 

Step 5: Apply the Tourniquet
   
  Just prior to blood collection, apply the tourniquet 4-6 inches from insertion site. The tourniquet should restrict venous flow only, can be easily released, and never tight enough to restrict the arterial blood flow. Never allow the tourniquet to be left on longer than two minutes. NEVER probe blindly in a patient's arm. Make only two attempts per person taking the blood.
   
   
 

Step 6: Skin Preparation
   
  Select the site of venipuncture. The practice of drawing blood for culture from catheters of the groin should never be performed when a peripheral (i.e., non-catheterized) site is available. Blood should be obtained from peripheral venous or arterial sites. Obtaining blood cultures from central venous catheters, arterial lines, and inguinal vessels increases the likelihood of obtaining a false positive blood culture.
   
 
   
  Palpate for the site from which to draw the sample. Obtain 1 Chloroprep-Frepp. Place the sponge down onto the arm in the area of the intended puncture site and prime the sponge by pressing the sponge up and down on the arm until the alcohol can be seen on the skin. Scrub the arm vigorously with the sponge for 30 seconds. Allow to air dry completely for 30 seconds
   
  DO NOT BLOW, FAN, OR WIPE THE AREA WITH GAUZE. DO NOT TOUCH THE AREA!
   
   
 

Step 7: BD Butterfly Method
   
  To be performed after inspecting bottles, disinfecting the tops, applying tourniquet, and skin preparation,:
   
 
   
1) Peel apart the package and remove blood collection set. Thread the Luer end of tubing set into Vacutainer holder. Remove sheath-covering needle at the wings.
   
 
   
2) Perform venipuncture.
   
 
   
3) Push and hold the Vacutainer holder over the top of the vial to puncture the septum. Collect blood in the aerobic bottle to the desired fill level on the vial. Inoculate the amount recommended on the bottle (8-10 cc for adults). The volume of the inoculum must never be less than 3.0 for the standard aerobic/F, standard anaerobic/F, aerobic plus, and anaerobic plus bottles. Pediatrics plus bottles will except 1-3 ml.
   
4) Monitor to ensure proper blood flow and fill level. Always keep the culture bottle sin the upright position while obtaining specimens.
   
5) Remove the holder from the vial. Immediately push and hold the holder onto the anaerobic bottle.
   
6) Collect blood to the desired fill level on the second vial. Remove the holder from vial. Note: If more samples are required, additional tubes may be drawn at this time using the Vacutainer holder. Follow the appropriate order of draw if further blood tests are required.
   
7) Removal-- When the final vial or tube is filled, activate the safety on the butterfly. Apply pressure to the insertion site. Only after bleeding has completely ceased apply a 2x2 gauze and tape or Band-Aid
   
8) Attach the patient label vertically (being careful not to completely cover the bottle barcode) to both blood culture bottles. These labels must include the patient's name, medical record number, room number, procedure ordered, collection date and time. The staff member who performed the venipuncture must also handwrite his/her initials, collection date and time, area of puncture site or if drawn from a central line on the patient's specimen label. All blood cultures are to be sent to the Laboratory within one hour.