Microbiology

There are several factors essential to providing high-quality microbiological testing. They are: proper collection of the specimen, proper labeling and rapid transport to the laboratory. All are extremely important. Failure to isolate the causative agent in a culture is frequently the result of faulty collecting or transport technique. The following sections outline important instructions that must be followed to ensure optimal recovery of pathogens.

Ordering Procedures

• The nurse or the physician enters microbiology orders into the computer system, where available.
• Requisition paperwork with complete information must accompany the specimen for delivery to the lab.
• The exact source of the specimen must be entered at the specimen prompt or listed on the requisition. If there is no code for the specimen type, a generic source is entered and the exact source must be noted on the requisition.
• The date and time of collection and the ordering physician is required.

SPECIMEN LABELING

  • Each specimen container must be labeled with patient’s full name, Social Security or medical record number, and date and time ofcollection. Unlabeled specimens will not be processed. The requisition is sent to the lab with the labeled specimen; it is not a substitute for labeling. The patient information on the requisition must match the label on the specimen.
  • All specimens must be placed in zip-close bags with the opening secure for transport to laboratory.

Requisition slips must be in the pouch outside of the bags.

DELIVERY OF SPECIMEN

  • Prompt delivery of specimens to the lab is essential if results of cultures are to be valid.

COLLECTION INSTRUCTIONS:

  • Whenever possible, specimens should be obtained prior to the administration of antibiotics. If a culture is taken after initiation of antibacterial therapy, the specific antibiotics given should be noted on the confirmation slip.
  • Cultures should be collected from the actual site of infection with as little external contamination as possible. This can be accomplished by cleaning surrounding area with 70% alcohol or sterile saline prior to collection of culture and by being careful to bypass areas of normal flora. Specimen should be submitted to lab in a sterile container or in appropriate transport medium as listed on Specimen Collection and Transport Chart.
  • Necessary collection supplies are available through the Courier Department. Certain items, such as culturette swabs and anaerobic collection tubes have an expiration date printed on the package. Do not use past the date of expiration.

CAUSES OF SPECIMEN REJECTION

In all cases of unacceptable specimens, the patient care unit must be notified and the nature of the discrepancy explained.

  • Discrepancy between patient identification on requisition and specimen container.
  • Unlabeled or improperly labeled specimen container.
  • Presence of gross external contamination on specimen container.
  • Exact specimen source not indicated on requisition slip.
  • Date and time of collection not indicated on requisition slip.
  • Specimen received on dried out swabs.
  • Urine held over 2 hours at room temperature.
  • Foley catheter tips are unacceptable due to skin flora contamination. Collect urine specimen before removing catheter or 2 hours after removal.
  • Specimen received in fixative.
  • Inadequate or improperly collected specimen.
  • Anaerobic culture from swabs of throat, nose, urethra, vagina, cervix, or rectum; expectorated sputum; voided or catheterized urine; or stool specimen.
  • Syringe with capped needle: it is against hospital policy to recap a needle. Contents of syringe can be transferred to a suitable sterile container. Alternatively syringe may be sent to lab after needle has been properly removed and disposed of and opening of syringe has been capped.
  • 24 hour urine or sputum collections for AFB or Fungus.
  • The microbiology lab will reject sputum based on their criteria. Notification of sputum rejection by the lab is a comment stating specimen is rejected, why, and call within 48 hours.
  • Stools collected in a non-screw top container (i.e stool in denture cups)
  • Only Liquid, unformed stool will be tested by microbiology. Formed stool samples will be canceled Only one (1) stool sample for difficile will be tested by microbiology during a 7 day period. Additional samples will be canceled. Formed stool will not be tested by microbiology. Regardless of result no further testing will be conducted within 7 days , after 7 days a 2nd specimen may be tested only if the clinical course of the patient changes.
  • MRSA by PCR specimens not collected using the appropriate Copan swab provided by the lab.

SPECIMEN TYPE

ANAEROBIC

Acceptable Specimens

  • Pus from closed abscess.
  • Pleural fluid by thoracentesis.
  • Urine by suprapubic bladder aspiration.
  • Pulmonary secretions by transtracheal aspiration.
  • Uterine secretions or sinus tract material by insertion of an intravenous type of catheter through a decontaminated area and aspiration with a syringe.
  • Unsuitable Specimens
    • Swabs from the throat, nose, ear, eye, decubiti, superficial wounds, urethra, vagina, cervix, or rectum.
    • Expectorated sputum, bronchial secretions, voided urine, feces, and gastric contents.
  • Methods of Collection
    • Anaerobic specimen collectors are provided by the laboratory throught the courier department. The collector transport system consists of a swab collector in a tube constructed so that once the specimen is collected, introduced into the smaller tube and the system activated, an anaerobic atmosphere is provided. Directions for the use of the system accompany each collector. The system can be used as a collector and transport system for specimen collected by swab, or as an anaerobic transport system for tissue, fluids, and other types of specimens. The activated collector should be held upright at all times and delivered to the laboratory ASAP. The anaerobic specimen collector is not intended for the collection of specimens suspected of containing Neisseria meningitidis or Neisseria gonorrhoeae.
    • Aspiration of fluid with sterile needle and syringe: After decontamination of the outer surface of the lesion, the fluid specimen should be aspirated with a sterile needle and syringe. Care should be taken to prevent air from entering the syringe. Once aspirated, the fluid may be transferred to the anaerobic collector, or the syringe needle may be removed and the opening plugged with a stopper. Transport immediately to the laboratory.

BLOOD

Bactec Aerobic, Anaerobic and Pediatric bottles are used in our laboratory and available through the courier department. Collect blood cultures as directed by the physician. If not otherwise specified, each set of blood cultures should be drawn at 15 minutes intervals. The maximum number of blood cultures per 24 hours per patient should not exceed 6 sets.

Adult patient: For each blood culture ordered, one Aerobic and one Anaerobic bottle should be drawn. Fill Aerobic bottle first. In the event that only one bottle can be obtained from a single venipuncture, the aerobic bottle must be drawn. Each set of blood cultures ordered must be obtained from a separate venipuncture site.

  • Aerobic bottle: fill with 3-10 mls of blood (10 mls optimal)
  • Anaerobic bottle: fill with 5-7 mls of blood
  • NOTE: It is very important to obtain optimal fill level of bottles to ensure recovery of pathogens. DO NOT underfill or overfill bottles.
  • Pediatric Patient: Draw 0.5-3 mls of blood into Pediatric Bactec bottle.
    • NOTE: Aerobic culture bottles contain resins to neutralize antibiotics. No special procedures are necessary when an ARD or Resin bottle is requested.
  • Fungus Blood cultures: Draw one Myco/F blood culture bottle for each order of blood for fungus. The length of the incubation period will be changed from 5 to 30 days in the Bactec instrument.
  • Mycobacteriology (AFB) Blood cultures: Draw one Myco/F blood culture bottle for each order of AFB blood.The order should be placed in the computer as an Mycobacterium blood culture (HAFBLD) with the specimen source as blood. The bottle should be sent to the lab as soon as possible but within 8 hours of collection. Maryview will send the bottle to Sentara Norfolk General lab for processing.

 CEREBROSPINAL FLUID

All CSF specimens are treated as STAT. Gram stains on all CSF’s except myelograms are required and therefore should be requested.

Lumbar spinal puncture is the procedure used by physicians to obtain cerebrospinal fluid for culture and other laboratory tests. Lumbar punctures must be performed under conditions of strict asepsis. Since contamination of the specimen can occur readily and confuse the identification of the etiologic agent, the skin should be disinfected properly prior to the puncture. Specimen should be collected by the physician into sterile screw cap tubes in order to preclude leakage and loss or contamination of the contents. The specimen should be delivered to the laboratory immediately and laboratory personnel should be notified of its arrival. If there is to be a delay in processing the specimen, the fluid should be left at room temperature. The Microbiology lab should process tube #2. All CSF samples sent for bacterial and/or cryptococcal antigen testing must have back up cultures ordered and performed.

EAR:

Material from the ear, especially that obtained after perforation of the eardrum, is usually collected by the physician, using sterile equipment and a sterile swab. In external otitis the external ear should be cleansed with suitable disinfectant in order to free the skin of contaminating bacterial flora before the culture is taken. A small swab is then carefully inserted into the ear canal and rotated to collect the exudate.

EYE:

The number of organisms recovered from cultures of many eye infections may be relatively low, due to the constant working activity of the tears and their antibacterial constituents. Swab specimens are often inadequate since the sample size is small. An additional problem is that topical anesthetics possess antimicrobial activity. It is therefore recommended that as much specimen as possible be obtained on the swab and that the specimen be collected before application of topical anesthetics. Care should be taken that eyelids and eyelashes not be touched during specimen collection. Some infections require collections of corneal scrapings as adequate specimen. All instruments used for collection of eye cultures must be sterile.

FECAL:

Stool specimens should be collected in a sterile screw top container and delivered to the lab as soon after collection as possible. Consider use of C. difficile toxin by PCR instead of cultures. Stools contaminated with urine, mineral oil or barium are unacceptable. If necessary a sterile swab may be used to obtain fecal samples for culture only. The swab should be passed beyond the anal sphincter, carefully rotated and withdrawn. Liquid stool specimens for parasitological exam must be delivered to lab and be placed in fixative within 1 hour of collection. Stool specimens submitted for C.difficile testing must be liquid or semi-formed, any formed stool will be rejected.

FLUID (OTHER THAN CSF):

The percutaneous aspiration of pleural, pericardial, peritoneal and synovial fluids must be performed aseptically to avoid contamination of the specimen and to prevent the accidental introduction of microorganisms into these anatomical spaces. The specimen should immediately be injected into a sterile tube, and/or into an anaerobic collector. Gram-stained smears of the centrifuged sediment of clear or slightly cloudy fluids should be made and examined carefully. Purulent fluids should be smeared directly and examined.

GENITAL (CERVICAL, VAGINAL, URETHRAL):

Using a sterile swab the specimen is obtained by collecting a small amount of exudate or pus from the infected area. Principal culture for venereal disease areas are vagina, cervix, urethra, and bartholin gland. Secondary sources for venereal diseases are rectum, nasopharynx, eye, and cutaneous lesions. In the female the best site from which to obtain a culture is the cervix. The cervical culture is obtained with the aid of a sterile speculum and an alginate or cotton-tipped swab. In situations where a cervical specimen is not indicated (children or hysterectomized patients) a urethral or vaginal culture may be substituted. In male patients with a purulent urethral exudate, the examination of a Gram-stained direct smear is usually sufficient to confirm a clinical diagnosis of gonorrhea. However, since an appreciable number of males may be asymptomatic, a urethral culture is recommended. The culture is obtained by gently inserting a thin alginate urethrogenital swab approximately 2 cm. into the urethra, gently rotating and removing it.

NASAL:

Nasal cultures can be obtained to demonstrate organisms such as Staphylococcus aureus in the nose that can be transmitted by hospital personnel to patients within the hospital. These are normally just to identify Staphylococcus carriers. Nose cultures may also be obtained to demonstrate the causative agent of sinusitis. A culturette swab should be entered in each nostril, rotated a few times, and then placed back in the culturette. Nasal Swabs for MRSA screen by PCR must be collected on Copan swab provided by lab available through the courier department and are only performed on nares sources.

SPUTUM:

The patient should be instructed to obtain material from deep cough (tracheobronchial) expectorated directly into a sterile container. l – 3 ml. of purulent or mucopurulent material is sufficient. Saliva is an unacceptable specimen. Sputum collected in alcohol can not be cultured.

THROAT:

Throat cultures are most frequently obtained for the diagnosis of streptococcal pharyngitis and less common for the diagnosis of pertussis, diphtheria, and pharyngitis due to gonococcus or viruses. The proper technique for collecting a throat culture is very important. A culturette swab should be used along with a tongue depressor. The tongue depressor is held in one hand and used to depress the tongue to minimize contamination of the swab with oral secretions which may dilute, overgrow or inhibit the growth of pharyngeal flora. Both tonsillar areas, the posterior pharynx, and any areas of inflammation, ulceration exudation or capsule formation should be swabbed vigorously.

URINE:

Collections, storage, and transport of urine specimens for bacteriological examination is of the utmost importance. Contaminating bacteria can multiply in specimens standing at room temperature and invalidate the results of both microscopic and culture examination of urine. Specimens not examined or cultured within 1 hour after voiding must be refrigerated. Urine specimens may be collected by diagnostic catheterization, by the clean voiding midstream technique, by suprapubic aspiration, or from an indwelling catheter. Method of collection should be noted in the specimen comment line of the request. It is best to obtain early morning specimens whenever possible. Obtaining a clean voided urine specimen will vary greatly depending on the age and sex and ability of the patient to cooperate. The following detailed instructions to lab staff, nursing personnel, and patients are recommended for collection of midstream specimens from patients.

Cleanse the periurethral area (tip of penis, labial folds, vulva) with two separate washes with plain soap and water or mild detergent. Rinse well with water to remove the detergent and with the glans penis or labial folds retracted, have the patient flush the

urethral passage with the first portion of voiding, which is discarded, collect the subsequent urine voided directly into a sterile container. 

WOUND:

The surface of cutaneous wounds or decubitus ulcers frequently is colonized with environmental bacterial and swab samples often do not reflect the true cause of the infectious process. For that reason, the most desirable method of collecting cutaneous specimens is by aspirating lobulated purulent material from the depths of the wound with sterile needle and syringe. The wound margins should be decontaminated as much as possible with surgical soap and application of 70% ethyl or isopropyl alcohol. If material is obtained in the syringe, the needle should be removed and opening of syringe should be plugged, or contents of the syringe can be placed into a sterile test tube for delivery to lab. If material cannot be obtained with a needle and syringe, then a swab must be used to collect the specimen, the wound margins should be gently separated and the tip of the swab extended deep into the wound, taking care not to touch the adjacent skin margins. All wound cultures should have gram stains performed routinely when proper amount of material or number of swabs is submitted.

REFERENCES:

  1. Murray, Baron, Jorgensen, Pfaller and Yolken, Manual of Clinical Microbiology, 8th   edition, pp. 288-307, 2003
  2. Bailey and Scott’s Diagnostic Microbiology; Forbes, Sahm, Weissfeld 12th edition, CV Mosby Elsevier pg62-77 2007.