Grandview Medical Center

1. Good Microbiology depends upon and begins with proper specimen collection and handling.  Evaluation of a specimen may be jeopardized if it has been obtained either with improper technique, in an inadequate amount, from an inappropriate site, or improperly preserved.  Specimens may be further compromised by improper or delayed transport to the laboratory resulting in quantitative distortion of the relationships among the organisms.  The requests and instructions contained in this guideline are designed to provide the laboratory with proper material to aid the physician in patient care.  ALL microbiology specimens collected shall be delivered to the laboratory as soon after collection as possible.
2. Gram stains, on all specimens other than sputum, shall be ordered as a separate order from the culture.  A gram stain is included in the order for a sputum culture.
3. The laboratory shall be notified of any suspected unusual pathogens requiring special isolation techniques or special media.
4. No specimen received for Microbiology testing can be rejected without considering the difficulty of collecting the specimen.  Prior to rejecting a specimen, the physician or nurse in charge will be phoned to discuss the questioned specimen.
5. If the specimen cannot be easily recollected, such as a specimen from a surgical procedure, the sub-optimal specimen will be processed with the appropriate message to facilitate the clinician’s interpretation of the test results.

 

REQUESTING THE CULTURE:

1. All cultures shall be performed at the order of a licensed physician.
2. The request form for the culture shall contain:
   1. The full name of the patient
   2. The location of the patient
   3. The ordering physician
   4. The source of the specimen
   5. The time and date of collection of the specimen
   6. The initials of the collector
   7. The type of culture requested  –  routine for site, anaerobic, acid fast, fungus
   8. Any comments that relate to the culture
3. The specimen shall be labeled with the following information:
   1. The full name of the patient
   2. The date of birth
   3. The source of the specimen
   4. The time and date of collection
   5. The initials of the collector

 

COLLECTION INFORMATION:

Collection of specimens for culture is performed in a manner to insure the results are accurate, meaningful, and appropriate for the treatment of infections.  Items to be considered during the collection of specimens for culture:

1. Usual flora of the collection/infection site
2. Contaminating organisms that may be present at the collection site
3. Aerophilic (aerobic or anaerobic) nature of the suspected organism/s
4. Specimen requirements for the site

 

COLLECTION/TRANSPORT INFORMATION BY CULTURE SITE:

The following will list the source of culture material, the proper collection device, the cleansing technique, the collection technique, the timing of collection,

 

 

BLOOD CULTURES:

1. Collection materials:
   1. 21g butterfly and blood collection cannula (Can use a 21g hypodermic needle and a syringe)
   2. Aerobic blood culture bottle
   3. Anaerobic blood culture bottle
   4. Alcohol prep
   5. Chlorhexadine gluconate (CHG) prep is preferred.  (Iodine may be used.)
   6. Gauze pads
2. Cleansing of the area
   1. Cleanse the tops of the culture bottles with an alcohol prep
   2. Clean the area to be stuck in a expanding circular motion with the alcohol prep
   3. Let dry
   4. Clean the area to be stuck using a CHG prep. Scrub in a vertical and horizontal pattern. If iodine is used, cleanse in an expanding circular motion with the iodine prep.
   5. Let dry
3. Collection of specimen
   1. Butterfly

i.    Insert the butterfly into the vein

ii.    Maintain the bottle below level of the needle

iii.    Insert the aerobic bottle

iv.    Let the bottle draw until no more blood is coming into the bottle

v.    Insert the anaerobic bottle

vi.    Let the bottle draw until no more blood is coming into the bottle

vii.    Collect any remaining labs that are to be drawn with this stick

1. Syringe

i.    Draw the syringe completely full

ii.    Using a transfer device, place one half the blood in the syringe into the anaerobic bottle (no less than 5 mL into each bottle)

iii.    Using the transfer device, place the rest of the blood in the syringe into the aerobic bottle

 

BLOOD VOLUMES TO BE COLLECTED FOR BLOOD CULTURES
AGE	VOLUME	INSTRUCTIONS
<3 years old	Draw approximately 1 ml for each year of life	Transfer the entire amount into a PEDIATRIC bottle
4 – 10 years old	Draw approximately 1 ml for each year of life	Transfer entire amount into an adult AEROBIC bottle
> 10 years old with good veins	Draw 20 ml	Place 10 ml into an AEROBIC bottle Place 10 ml into an ANEROBIC bottle
> 10 years old with poor veins	10 – 20 ml obtained	Place 10 ml into an AEROBIC bottle and the remaining into an ANEROBIC bottle
	Less than 10 ml obtained	Place entire amount into an AEROBIC bottle
All others	The quantity the bottle will draw	Draw using a butterfly directly into the AEROBIC bottle then into the ANEROBIC bottle until the blood stops flowing

 

1. Preservation of specimen
   1. Send to the laboratory as soon as possible after collection
   2. Do not refrigerate
   3. Do not incubate

 

1. Collection timing
   1. Collect before starting antibiotics
   2. Collect two sets with 30 minutes between each set or each set from a different collection site
   3. If intermittent bacteremia is suspected, collect the blood cultures 1 hour before anticipated spike of fever
   4. As ordered by the physician

 

CSF / BODY FLUIDS:

1. Collection devices
   1. Spinal tap tray
   2. Sterile plastic collection tubes (these are within the spinal tap tray)
2. Cleansing of the area
   1. Cleanse the area of the tap with iodine solution or CHG solution
3. Collection of specimen
   1. Spinal fluid

i.    Collect 2-3 ml spinal fluid as it come out of the spinal tap needle

ii.    If tuberculosis is suspected, collect 5-8 ml of spinal fluid

iii.    Divide the collection among three tubes

iv.    Label the tube in the order of collection

1. Other body fluids

i.    If anaerobic bacteria is suspected, collection into a closed container that prevents contamination with air

1. Preservation of specimen
   1. Send to the laboratory as soon as possible after collection
   2. Do not refrigerate
2. Collection timing
   1. Collect before starting antibiotics
3. Ordering
   1. Order all the tests needed

i.    Cell count with/without diff

ii.    Chemistries

iii.    Culture aerobic  (Use boldy fluid or CSF culture)

iv.    Culture anaerobic

v.    Culture AFB

vi.    Gram stain

vii.    State fluid type in the “comments” section

 

EYE / ROUTINE:

1. Collection devices
   1. Swab with transport media
2. Cleansing of the area
   1. No special cleansing required
3. Collection of specimen
   1. Pull the lower lid down and collect the discharge
4. Preservation of specimen
   1. Send to the laboratory as soon as possible after collection
   2. Do not refrigerate
   3. Make sure the swab is into the preservative
5. Collection timing
   1. No special timing required

 

EYE / DNA PROBE:

1. Collection devices
   1. GEN probe device for GC or Chlamydia
2. Cleansing of the area
   1. No special cleansing required
3. Collection of specimen
   1. Pull down the lower lid and collect the specimen from within the eye
   2. Place the swab into the container making sure the swab is into the preservative
4. Preservation of specimen
   1. Send to the laboratory as soon as possible after collection
5. Collection timing
   1. No special timing required

FUNGUS:

1. Collection devices
   1. Swab with transport media
   2. Sterile container
2. Cleansing of the area
   1. Cleanse the area with alcohol and let dry
3. Collection of specimen
   1. Rub the swab over the area
   2. Using a sharp blade, scrap the area around the perimeter of the lesion. Place into a sterile container
   3. Clippings of nails.  Place into a sterile container.
4. Preservation of specimen
   1. Send to the laboratory as soon as possible after collection
5. Collection timing
   1. No special timing required

 

GASTRIC OCCULT BLOOD:

1. Collection devices
   1. Gastric occult blood card
2. Cleansing of the area
   1. No cleansing required
3. Collection of specimen
   1. Place parts of the sample that appears to contain blood onto the gastric occult blood card
4. Preservation of specimen
   1. Send to the laboratory as soon as possible after collection
5. Collection timing
   1. No special timing required

 

GENITAL CULTURE:

1. Collection devices
   1. Swab with transport medium
2. Cleansing of the area
   1. No special cleansing indicated
3. Collection of specimen
   1. Male

i.    Collect any discharge onto the swab

1. Female

i.    Collect discharge from the cervix

1. Preservation of specimen
   1. Send to the laboratory as soon after collection as possible
   2. Make sure the swab in into the preservative
   3. Do not refrigerate
2. Collection timing
   1. Collect before starting antibiotics

 

GENITAL DNA PROBE:

1. Collection devices
   1. GEN-probe device for the organism suspected (GC or Chlamydia) for male or female
   2. Use ONLY the swabs provided within the collection kit
2. Cleansing of the area
   1. Male:

i.    Patient should not have urinated for at least 1 hour prior to sampling

 

1. Female:

i.    Remove the excess mucus from the cervical os and surrounding mucosa

1. Collection of specimen
   1. Male

i.    Insert the swab from the collection kit 2-4 cm into the urethra

ii.    Rotate clockwise for 2-3 seconds

iii.    Withdraw the swab

iv.    Place the swab into the transport media and break off the excess shaft

1. Female

i.    Insert swab from the collection kit 1-1.5 cm into the endocervical canal

ii.    Rotate the swab clockwise for 10-30 seconds in the endocervical canal

iii.    Avoid contact with vaginal mucosa

iv.    Place the swab into the transport media and break off the excess shaft

1. Preservation of specimen
   1. Send to the laboratory as soon as possible after collection
2. Collection timing
   1. No special timing required

 

MYCOBACTERIOLOGICAL (AFB):

1. SPUTUM
   1. See instructions below within “Sputum / AFB / Mycobacteriology”
2. BRONCHIAL WASHING
   1. Collect in a sterile container which will hold at least twice the volume of the specimen
3. GASTRIC WASHING
   1. Collect the early morning specimen (before breakfast) into a sterile collection container
4. URINE
   1. Collect the first morning specimen in a sterile container.
   2. Must be at least 20 ml
   3. Collect three consecutive morning specimens
   4. DO NOT collect a 24-hour urine
5. OTHER
   1. Collect into a sterile container

 

NASAL:

1. Collection devices
   1. Swab containing transport medium
2. Cleansing of the area
   1. No special cleansing required
3. Collection of specimen
   1. Insert the swab into the nares until resistance is felt
   2. Rotate the swab
   3. Insert the swab into the transport tube into the preservative
4. Preservation of specimen
   1. Make sure the swab is into the transport media
   2. If the transport media is in a capsule, crush the capsule
5. Collection timing
   1. No special timing required

 

NASOPHARYNGEAL:

1. Collection devices
   1. Nasopharyngeal swab
2. Cleansing of the area
   1. No special cleansing of the area required
3. Collection of specimen
   1. Insert the swab into the nose until it is passed the nasal area and into the nasopharyngeal area
   2. Let the swab remain a few seconds
   3. Place the swab into the tube and into the transport medium
4. Preservation of specimen
   1. Insert the swab into the tube with the transport medium
5. Collection timing
   1. No special timing required

 

SPUTUM / ROUTINE CULTURE: (Gram stain is performed on all sputum specimens with or without a specific order for gram stain.)

1. Collection devices
   1. Sterile collection cup
2. Cleansing of the area
   1. No cleansing of the area required
3. Collection of specimen
   1. The first morning specimen is the best
   2. The specimen must come from within the lungs or airway not the nasopharyngeal area
4. Preservation of specimen
   1. Send to the laboratory as soon after collection as possible
5. Collection timing
   1. Before antibiotics started

 

SPUTUM / AFB / MYCOBACTERIOLOGY:

1. Collection devices
   1. Sterile cup
2. Cleansing of the area
   1. No special cleansing required
3. Collection of specimen
   1. The first morning specimen is best
   2. The specimen must come from within the lungs or airway not the nasopharyngeal area
4. Preservation of specimen
   1. Send to the laboratory as soon as possible
5. Collection timing
   1. Sputum for AFB is usually ordered as three specimens.  At least one specimen must be an early morning specimen.  The other two can be collected 8 hours apart.

 

STOOL / ROUTINE CULTURE / CLOSTRIDIUM DIFFICILE TOXIN A & B/O&P:

1. Collection devices
   1. Collection container that will allow the collection of the stool with no contamination by urine
   2. Swab may be used
2. Cleansing of the area
   1. No special cleansing required
3. Collection of specimen
   1. Collect the specimen into a container with no contamination by urine
   2. Place the specimen into a sterile cup.  The minimum for each test is listed below.  If more than one of the tests, please submit the total volume for all the tests ordered.

i.    Minimum volume for C. diff. is 0.5 mL

ii.    Minimum volume for O&P is 5 mL

iii.    Minimum volume for stool WBC is 2.5 mL

iv.    Minimum volume for Fecal Fat is 1 mL

v.    Minimum volume for Helicobacter pylori stool antigen is 1 mL

1. Preservation of specimen
   1. Send to the laboratory as soon as possible after collection
   2. Stool for culture only shall be refrigerated if not plated within 1 hour
2. Collection timing
   1. No special timing required

 

STOOL / OCCULT BLOOD:

1. Collection devices
   1. Container that will allow the collection of the stool with no contamination by urine
2. Cleansing of the area
   1. No special cleansing required
3. Collection of specimen
   1. Collect the stool with no contamination by urine
   2. Place a part of the specimen that appears to contain blood onto a fecal occult blood card
4. Preservation of specimen
   1. Send to the laboratory as soon as possible after collection
5. Collection timing
   1. No special timing required

 

THROAT STREP A SCREENS (QUICK STREP):

1. Must use a swab provided with the testing kit (contact lab for this swab)
2. Collect a routine culturette for culture if the Strep A Screen is negative

 

THROAT:

1. Collection device
   1. Sterile swab with transport media to preserve the aerobic organisms
2. Cleansing of the area
   1. No special cleaning required
3. Collection of specimen
   1. Swab the back of the throat being sure to swab any visible lesions
   2. Do not swab the mouth region
   3. Place the swab into the tube and into the preservative
4. Preservation of specimen
   1. Send to the laboratory as soon as possible after collection
5. Collection timing
   1. No special timing required

 

URINE:

1. Collection devices
   1. Refer to the procedure “Mid-stream Clean Catch Urine Collection Instructions”
   2. If collecting from a Foley catheter, collect using a syringe with access device from the drainage line not the bag
2. Cleansing of the area
   1. Refer to the procedure “Mid-stream Clean Catch Urine Collection Instructions”
   2. Cleanse the access port with alcohol prep and let dry
3. Collection of specimen
   1. Refer to the procedure “Mid-stream Clean Catch Urine Collection Instructions”
   2. If collecting from a Foley catheter, use a syringe to draw from the collection port
   3. NEVER collect a specimen from the collection bag
4. Preservation of specimen
   1. Send to the laboratory as soon as possible after collection
   2. Refrigerate
5. Collection timing
   1. Before antibiotics are started
   2. The first morning specimen is the best

VIRAL:

1. Collection devices
   1. Viral collection and transport device
2. Cleansing of the area
   1. Cleanse the area with sterile water

 

1. Collection of specimen
   1. Swab

i.    Rub the lesion with the swab

1. Tissue/scrapings

i.    Excise the tissue or scrape the lesion with a sterile blade

1. Feces

i.    Use a pea-sized portion of the specimen

1. Preservation of specimen
   1. Place the swab into the transport media breaking off the excess shaft
   2. Place the tissue/scrapings into the transport media
   3. Place the pea-sized portion of the stool specimen into the transport media
2. Collection timing
   1. No special timing required

 

WOUND / SUPERFICIAL:

1. Collection device
   1. Sterile culturette with media to preserve aerobic and anaerobic organisms
   2. One culturette for aerobic and one culturette for anaerobic
2. Cleansing the area
   1. Alcohol prep
3. Collection of the specimen
   1. Collect the drainage
   2. Place the swab into the container and into the preservative
4. Preservation of the specimen
   1. Send to the laboratory as soon as possible after collection
5. Collection timing
   1. No special timing required
6. Order
   1. Wound culture
   2. Anaerobic culture if desired
   3. Gram stain if desired
   4. Specify location

 

WOUND / DEEP:

1. Collection device
   1. Sterile culturette with media to preserve aerobic and anaerobic organisms
   2. One culturette for aerobic and one culturette for anaerobic
   3. Syringe with needle
2. Cleansing the area
   1. Alcohol prep
3. Collection of the specimen
   1. Collect the specimen from deep within the tissue by expressing the drainage and collecting onto a swab or aspiration with a needle and syringe
   2. Place the swab into the container and into the preservative
4. Preservation of the specimen
   1. Send to the laboratory as soon as possible
5. Collection timing
   1. No special timing required
6. Order
   1. Wound culture
   2. Anareobic culture if desired
   3. Gram stain if desired
   4. Specify location

 

WOUND / DECUBITUS ULCER:

1. Collection device
   1. Sterile needle and syringe
   2. Culturette with transport media
   3. One culturette for aerobic and one culturette for anaerobic
2. Cleansing the area
   1. Remove the decomposing tissue
3. Collection of the specimen
   1. Aspirate fluid from deep within the decubitus
   2. Using the culturette, obtain the specimen from the base of the ulcer
   3. Place the culturette into the container and into the preservative
4. Preservation of the specimen
   1. Send to the laboratory as soon as possible after collection
5. Collection timing
   1. No special timing required
6. Order
   1. Wound culture
   2. Anaerobic culture if desired
   3. Gram stain if desired
   4. Specify location

 

WOUND / INTRAVENOUS OR INTRA-ARTERIAL CATHETER:

1. Collection device
   1. Sterile culturette with transport media to preserve the aerobic and anaerobic organisms
   2. One culturette for aerobic and one culturette for anaerobic
2. Cleansing the area
   1. Alcohol prep
3. Collection of the specimen
   1. Collect any discharge from the site
   2. Site may be compressed to initiate discharge
   3. Place the swab into the container and into the preservative
4. Preservation of the specimen
   1. Send to the laboratory as soon as possible after collection
5. Collection timing
   1. No special timing required
6. Order
   1. Wound culture
   2. Anaerobic culture if desired
   3. Gram stain if desired
   4. Specify location

 

INAPPROPRIATE SPECIMENS: (These absolutely CANNOT be used for testing.)

1. A smear on a microscope slide with a request for culture
2. A smear on a microscope slide received with a request for wet prep
3. Specimen dried out (stool, sputum) with a request for culture
4. Dry swab received for Trichomonas examination
5. Foley catheter tip received with request for culture
6. Specimen received in fixative with request for culture
7. Frozen specimen with request for wet prep or ova and parasites
8. Specimens transported in same bag with leakage causing gross contamination of all specimens
9. Specimen received in stool culture transport media with a request for ova and parasites
10. Stool with barium received with a request for ova and parasites
11. DNA probe analysis swab received with a request for culture
12. Routine culturette received with a request for Chlamydia or Neisseria gonorrhoeae by DNA probe
13. Routine culturette received with a request for Chlamydia culture
14. Specimen received in stool culture transport media with a request for occult blood
15. Specimens received in viral transport media with a request for bacterial culture
16. Specimen received in O & P transport kit with a request for fecal fat
17. Laboratory received an empty specimen container
18. Blood received in tube with ACD solution with request for blood culture
19. Two or more unlabeled specimens received together with requisitions for different patients
20. Use of a wooden swab instead of the swab provided for GC/Chlamydia DNA probe

 

SUB-OPTIMAL SPECIMENS: (These specimens will be tested with appropriate messages to the physician.)

1. Non-sterile container received with a request for culture
2. Sub-optimal specimen for the test requested
3. Specimen received in expired transport media with a request for culture
4. Sputum specimens containing greater than 25 epithelial cells on a gram stain examination with a request for culture
5. Dry swab received with a request for culture